Transition metal complexes of a bis[thio-hydrazide amide] compound

ABSTRACT

The present invention is directed to a compound comprising a bis[thiohydrazide amide] or a deprotonated form thereof, complexed to a transition metal cation, wherein the bis[thiohydrazide amide] is represented by Structural Formula (I): or a prodrug, isomer, ester, salt, hydrate, solvate, polymorph or a deprotonated form thereof. The present invention also provides a pharmaceutical composition comprising a compound of the invention and method of use thereof.

RELATED APPLICATIONS

This application is the National Stage filed under 35 USC 371 ofPCT/US2009/061491, filed Oct. 21, 2009, which claims the benefit of U.S.Provisional Application No. 61/196,943, filed Oct. 22, 2008 and isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

It has been reported in U.S. Pat. Nos. 6,800,660; 6,762,204; 7,037,940;7,001,923; and 6,924,312 that certain bis [thio-hydrazide amide]compounds significantly enhance the anti-cancer activity of paclitaxeland paclitaxel analogs. In particular,N-malonyl-bis(N′-methyl-N′-thiobenzoylhydrazide) in combination withpaclitaxel has been shown to increase the time to progression ofpatients suffering from stage IV metastatic melanoma relative topatients treated with paclitaxel alone. It would be advantageous to havestill more active bis[thiohydrazide amide] anti-cancer compounds.

SUMMARY OF THE INVENTION

The present invention is directed to a compound comprising abis[thio-hydrazide amide] represented by Structural Formula (I):

or a prodrug, isomer, ester, salt, hydrate, solvate, polymorph ordeprotonated form thereof complexed, coordinated or chelated to atransition metal cation. One example of a compound of this type isrepresented by Structural Formula (II):

or, a prodrug, isomer, ester, salt, hydrate, solvate, or polymorphthereof, wherein X is a transition metal cation having a +2 charge.

Another embodiment is a pharmaceutical composition comprising a compoundof the invention and a pharmaceutically acceptable carrier or diluent.The pharmaceutical compositions can be used in therapy, for example, asan anti-proliferative agent (e.g., anti-cancer agent). In addition, thepharmaceutical compositions can be used in therapy to treat disordersresponsive to Hsp70 induction, or the pharmaceutical compositions can beused in therapy to treat disorders responsive to natural killer cellinduction, such as bacterial infections, fungal infections, viralinfections, or parasitic infections. The pharmaceutical compositions canalso be used in therapy to treat, reduce or inhibit angiogenesis in asubject in need thereof.

The present invention also provides for a method of treating a subjectwith cancer, treating a subject with an Hsp70-responsive disorder,treating a subject with a disorder responsive to natural killer cellinduction or treating, reducing or inhibiting angiogenesis in a subjectin need thereof. The method comprises administering to the subject aneffective amount of a compound of the invention or a pharmaceuticalcomposition of the invention. In one embodiment, the compound of theinvention is administered with paclitaxel (Taxol®) or a paclitaxelanalog.

The use of a compound of the invention for the manufacture of amedicament for treating a subject with cancer, for treating a subjectwith an Hsp70-responsive disorder, for treating a subject with adisorder responsive to natural killer cell induction or for treating,reducing or inhibiting angiogenesis in a subject in need thereof is alsoprovided in the present invention.

The present invention is also directed to the use of a compound of theinvention for treating a subject with cancer, for treating a subjectwith an Hsp70-responsive disorder, for treating a subject with adisorder responsive to natural killer cell induction or for treating,reducing or inhibiting angiogenesis in a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the structure of paclitaxel (Taxol®).

FIG. 2 is the structure of docetaxel (Taxotere®).

FIGS. 3-23 are each the structure of a paclitaxel analog.

FIG. 24 is the structure of a polymer comprising a paclitaxel analoggroup pendent from the polymer backbone The polymer is a terpolymer ofthe three monomer units shown.

FIG. 25 shows the cytotoxicity of compounds 1 and 2 in confluent M14cells.

FIG. 26 is an ORTEP diagram of compound 1 showing 50% thermalellipsoids.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to transition metal complexes(coordinates or chelates) of a bis[thiohydrazide amide] represented byStructural Formula (I) or a prodrug, isomer, ester, salt, hydrate,solvate, polymorph or deprotonated form thereof. One example of acomplex of this type is represented by Structural Formula (II) describedabove.

As used herein, “complexed” means that the bis[thiohydrazide amide ] ora prodrug, isomer, ester, salt, hydrate, solvate, polymorph ordeprotonated form thereof attaches to the transition metal ion throughone or more coordinate covalent bonds or coordination bonds.

As used herein, “chelated” means that the bis[thiohydrazide amide] or aprodrug, isomer, ester, salt, hydrate, solvate, polymorph ordeprotonated form thereof binds to the transition metal ion at two ormore attachment points through coordinate covalent bonds or coordinationbonds.

As used herein, “coordinate”, “coordinated”, “coordinate covalent bond”and “coordination bond” have the meanings that are commonly known to oneof ordinary skill in the art.

As used herein, a “deprotonated form” of bis[thiohydrazide amide] refersto a molecule wherein one or more protons from the bis[thiohydrazideamide] or a prodrug, isomer, ester, salt, hydrate, solvate or polymorphthereof were removed. For example, a deprotonated form of thebis[thiohydrazide amide] of Structural Formula (I) is represented by thefollowing structural formula:

A “transition metal cation” refers to a positively charged ion of ametal in Groups 3-12 of the Periodic Table. Examples include Ni²⁺, Cu⁺,Cu²⁺, Co²⁺, Co³⁺, Fe²⁺, Fe³⁺, Zn²⁺, Pt²⁺, Pd²⁺, V⁴⁺, V⁵⁺, Cr²⁺, Cr³⁺,Cr⁴⁺, Mn²⁺, Mn³⁺, Mn⁴⁺, and Mn⁵⁺. In a specific embodiment, thetransition metal cations have a +2 charge. Examples include Ni²⁺, Cu²⁺,Co²⁺, Fe²⁺, Zn²⁺, Pt²⁺ and Pd²⁺. In a specific embodiment, thetransition metal cation is Cu⁺, Cu²⁺ or Ni²⁺. In a more specificembodiment, the transition metal cation is Cu²⁺. The molar ratio ofbis[thiohydrazide amide] or a prodrug, isomer, ester, salt, hydrate,solvate, polymorph or a deprotonated form thereof to transition metalcation recited in this paragraph is, for example, equal to or greaterthan 0.5 and equal to or less than 2.0 (i.e. 0.5≦ratio≦2.0) or 1:1.

Exemplary compounds of the invention include Compound 1 and Compound 2:

or a prodrug, isomer, ester, salt, hydrate, solvate or polymorphthereof.

The compounds of the invention are advantageously in substantially pureform, e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, 99.5%or 99.9% pure by weight. “Percent purity by weight” means the weight ofthe compound divided by the weight of the compound plus impurities times100%.

The compounds of the present invention can be prepared by reacting thebis[thiohydrazide amide] represented by Structural Formula (I) or aprodrug, isomer, ester, salt, hydrate, solvate, polymorph or adeprotonated form thereof with a transition metal salt. The transitionmetal salt can be any inorganic or organic salts of the transition metalcation. For example, chloride salt, nitrate salt, sulfate salt, acetatesalt and the like can be reacted with a bis[thiohydrazide amide ] or aprodrug, isomer, ester, salt, hydrate, solvate, polymorph or adeprotonated form thereof described herein to afford the compounds ofthe present invention. In one embodiment, the transition metal salt is acopper(II) salt, such as CuCl₂. In another embodiment, the transitionmetal salt is a nickel(II) salt, such as NiCl₂.6H₂O.

The ratio of the bis[thiohydrazide amide] and the transition metalcation source used is typically in the range of 0.5 to 2.0 or 0.8 to1.2. In one embodiment, the ratio is about 1.

Solvents, such as methylene chloride, acetonitrile, acetone, alcohols(such as methanol, ethanol) tetrahydrofuran and water can be used in thereaction of the bis[thiohydrazide amide] derivative with the transitionmetal salts. In one embodiment, the solvent is ethanol.

The bis[thiohydrazide amides] used to prepare the disclosed compoundscan be prepared according to methods described in U.S. Pat. Nos.6,800,660, 6,762,204, and 6,825,235, and U.S. Publication No.2008/0146842. The entire teachings of these patents and publications areincorporated herein by reference.

Certain compounds of the invention may be obtained as different isomers(e.g., stereoisomers, coordination isomers, linkage isomers, hydrateisomers, and the like). The invention includes isomeric forms of thedisclosed compounds and both pure isomers and mixtures thereof,including racemic mixtures. Isomers can be separated and isolated usingany suitable method, such as chromatography.

The compounds of the invention may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asdouble-bond isomers geometric isomers), enantiomers, or diastereomers.According to this invention, the chemical structures depicted herein,including the compounds of this invention, encompass all of thecorresponding compounds' enantiomers, diastereomers and geometricisomers, that is, both the stereochemically pure form (e.g.,geometrically pure, enantiomcrically pure, or diastereomerically pure)and isomeric mixtures e.g., enantiomeric, diastereomeric and geometricisomeric mixtures). In some cases, one enantiomer, diastereomer orgeometric isomer will possess superior activity or an improved toxicityor kinetic profile compared to other isomers. In those cases, suchenantiomers, diastereomers and geometric isomers of compounds of thisinvention are preferred.

As used herein, the term “polymorph” means solid crystalline forms of acompound of the present invention described herein. Different polymorphsof the same compound can exhibit different physical, chemical and/orspectroscopic properties. Different physical properties include, but arenot limited to stability (e.g., to heat or light), compressibility anddensity (important in formulation and product manufacturing), anddissolution rates (which can affect bioavailability). Differences instability can result from changes in chemical reactivity (e.g.,differential oxidation, such that a dosage form discolors more rapidlywhen comprised of one polymorph than when comprised of anotherpolymorph) or mechanical characteristics (e.g., tablets crumble onstorage as a kinetically favored polymorph converts to thermodynamicallymore stable polymorph) or both (e.g., tablets of one polymorph are moresusceptible to breakdown at high humidity). Different physicalproperties of polymorphs can affect their processing. For example, onepolymorph might be more likely to form solvates or might be moredifficult to filter or wash free of impurities than another due to, forexample, the shape or size distribution of particles of it.

As used herein, the term “solvate” means a compound of the presentinvention described herein, that further includes a stoichiometric ornon-stoichiometric amount of solvent bound by non-covalentintermolecular forces.

As used herein, the term “hydrate” means a compound of the presentinvention described herein, that further includes a stoichiometric ornon-stoichiometric amount of water bound by non-covalent intermolecularforces.

As used herein, the term “prodrug” means a derivative of a compound thatcan hydrolyze, oxidize, or otherwise react under biological conditions(in vitro or in vivo) to provide a compound of this invention. Prodrugsmay become active upon such reaction under biological conditions, orthey may have activity in their unreacted forms.

The compounds of the invention or bis[thiohydrazide amide] describedherein may be present in the form of salts. In one embodiment, the saltsof the compounds of the invention refer to non-toxic “pharmaceuticallyacceptable salts.” In another embodiment, the salts also includenon-pharmaceutically acceptable salts, such as trifluoroacetate.

Pharmaceutically acceptable salt forms include pharmaceuticallyacceptable acidic/anionic or basic/cationic salts.

Pharmaceutically acceptable acidic/anionic salts include, the acetate,benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calciumedetate, camsylate, carbonate, chloride, citrate, dihydrochloride,edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate,glutamate, glycollylarsanilate, hexylresoreinate, hydrobromide,hydrochloride, hydroxynaphthoate, isethionate, lactate, lactobionate,malate, maleate, mandelate, mesylate, methylsulfate, mueate, napsylate,nitrate, pamoate, pantothenate, phosphate/diphospate, polygalacturonate,salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate,teoclate, tosylate, and triethiodide salts.

The compounds of the invention include pharmaceutically acceptableanionic salt forms, wherein the anionic salts include the acetate,benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calciumedetate, camsylate, carbonate, chloride, citrate, dihydrochloride,edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,lactobionate, malate, maleate, mandelate, mesylate, methylsulfate,mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphospate,polygalaeturonate, salicylate, stearate, subacetate, succinate, sulfate,tannate, tartrate, teoclate, tosylate, and triethiodide salts.

One embodiment of the present invention is a method of treating asubject with a proliferative disorder comprising administering to thesubject an effective amount of a compound or a pharmaceuticalcomposition described herein. Cancer, including multidrug resistantcancer, is one type of proliferative disorder that can he treated withthe compounds of the invention. Non-malignant proliferative disordersare also included in the invention.

“Treating a subject with a cancer” includes achieving, partially orsubstantially, one or more of the following: arresting the growth orspread of a cancer, reducing the extent of a cancer (e.g., reducing sizeof a tumor or reducing the number of affected sites), inhibiting thegrowth rate of a cancer, ameliorating or improving a clinical symptom orindicator associated with a cancer (such as tissue or serum components)and/or reducing the likelihood of the cancer recurring once it has beenremoved or gone into remission.

It has been surprisingly found that the transition metal chelate,coordinate or complex compounds disclosed herein show sufficientanti-cancer activity to make them suitable for monotherapies, as well asin combination or in co-therapies with other anti-proliferative oranticancer therapies. In particular, it has been found that transitionmetal chelates, coordinates or complexes ofN-malonyl-bis(N′-methyl-N′-thiobenzoylhydrazide) or a prodrug, isomer,ester, salt, hydrate, solvate, polymorph or deprotonated form thereof,can have sufficient anti-cancer activity to make them suitable formonotherapies, as well as in combination or in co-therapies with otheranti-proliferative or anticancer therapies such as paclitaxel.

Other anti-proliferative or anticancer therapies may be combined hecompounds or the pharmaceutical compositions of this invention to treatproliferative diseases and cancer. Examples include combination therapywith other anti-cancer drugs, surgery, radiotherapy (including, but notlimited to, gamma-radiation, neutron beam radiotherapy, electron beamradiotherapy, proton therapy, brachytherapy, and systemic radioactiveisotopes), thermal therapy (see, for example, U.S. Publication No.2008/0119440, the entire teachings of which are incorporated herein byreference) and endocrine therapy. Other anticancer agents that may beused in combination with the compounds or the pharmaceuticalcompositions of the present invention include biologic responsemodifiers (including, but not limited to, interferons, interleukins, andtumor necrosis factor (TNF)), hyperthermia and eryotherapy, agents toattenuate any adverse effects (e.g., antiemetics), and other approvedchemotherapeutic drugs. Specific examples of anticancer agents aredescribed in detail below. Preferably, the co-administered anti-cancerdrug is an agent that stabilizes microtubules, such as paclitaxel or ananalog of paclitaxel.

Cancers that can be treated or prevented by the methods of the presentinvention include, but are not limited to human sarcomas and carcinomas,e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastotna,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myeloeytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrobm's macroglobulinemia, and heavychain disease.

Other examples of leukemias include acute and/or chronic leukemias,e,g., lymphocytic leukemia e.g., as exemplified by the p388 (murine)line), large granular lymphocytic leukemia, and lymphoblastic leukemia;T-cell leukemias, e.g., T-cell leukemia (e.g., as exemplified by theCEM, Jurkat, and HSB-2 (acute), YAC-1(murine) cell lines), T-lymphoeyticleukemia, and T-Iymphoblastic leukemia; B cell leukemia (e.g., asexemplified by the SB (acute) cell line), and B-lymphocytic leukemia;mixed cell leukemias, e.g., B and I cell leukemia and B and Tlymphocytic leukemia; myeloid leukemias, e.g., granulocytic leukemia,myeloeytic leukemia e.g., as exemplified by the HL-60 (promyelocyte)cell line), and myelogenous leukemia (e.g., as exemplified by the K562(chronic) cell line); neutrophilic leukemia; eosinophilic leukemia;monocytic leukemia (e.g., as exemplified by the THP-1(acute) cell line);myelomonocytic leukemia; Naegeli-type myeloid leukemia; andnonlymphocytic leukemia. Other examples of leukemias are described inChapter 60 of The Chemotherapy Sourcebook, Michael C. Perry Ed.,Williams & Williams (1992) and Section 36 of Holland Frie CancerMedicine 5th Ed, Bast et. al. Eds., B.C. Decker Inc. (2000). The entireteachings of the preceding references are incorporated herein byreference.

Additional cancers that can be treated or prevented by the methods ofthe present invention include, but are not limited to oral cavity andpharynx cancers, including tongue, mouth, pharynx, and other oral cavitycancers; digestive system cancers, including esophagus, small intestine,rectum, anus, anal canal, anorectum, liver and intrahepatic bile duct,gallbladder and other biliary, pancreas and other digestive organs;respiratory system cancers, including larynx and bronchus; bone andjoint cancers; soft tissue (including heart) cancers; genital systemcancers, including uterine cervix, uterine corpus, ovary, vulva, vaginaand other genital, female, testis, penis and other genital, male;urinary system cancers, including kidney and renal pelvis, and ureterand other urinary organs; eye and orbit cancers; leukemia, includingacute myeloid leukemia and chronic myeloid leukemia.

In one embodiment, the disclosed method is believed to be particularlyeffective in treating a subject with non-solid tumors such as multiplemyeloma. In another embodiment, the disclosed method is believed to beparticularly effective against T-leukemia (e.g., as exemplified byJurkat and CEM cell lines); B-leukemia (e.g., as exemplified by the SBcell line); promyeloeytes e.g., as exemplified by the HL-60 cell line);uterine sarcoma (e.g., as exemplified by the MES-SA cell l(ne);monocytic leukemia (e.g., as exemplified by the THP-1(acute) cell line);and lymphoma e.g., as exemplified by the U937 cell line).

In another embodiment, the disclosd method is believed to beparticularly effective in treating a subject with melanoma.

In another embodiment, the disclosed method is believed to beparticularly effective in treating a subject with renal cell carcinoma.

The disclosed method is particularly effective at treating subjectswhose cancer has become “drug resistant”. A cancer which initiallyresponded to an anti-cancer drug becomes resistant to the anti-cancerdrug when the anti-cancer drug is no longer effective in treating thesubject with the cancer. For example, many tumors will initially respondto treatment with an anti-cancer drug by decreasing in size or evengoing into remission, only to develop resistance to the drug. Drugresistant tumors are characterized by a resumption of their growthand/or reappearance after having seemingly gone into remission, despitethe administration of increased dosages of the anti-cancer drug. Cancersthat have developed resistance to two or more anti-cancer drugs are saidto be “multi-drug resistant”. For example, it is common for cancers tobecome resistant to three or more anti-cancer agents, often five or moreanti-cancer agents and at times ten or more anti-cancer agents.

Numerous non-cancer diseases involve excessive or hyperproliferativecell growth, termed hyperplasia. As used herein, the terms“proliferative disorder”, “hyperproliferative disorder,” and “cellproliferation disorder” are used interchangeably to mean a disease ormedical condition involving pathological growth of cells. Such disordersinclude cancer.

Non-cancerous proliferative disorders include smooth muscle cellprolrferation, systemic sclerosis, cirrhosis of the liver, adultrespiratory distress syndrome, idiopathic, cardiomyopathy, lupuserythematosus, retinopathy, e.g., diabeticretinopathy or otherretinopathies, cardiac hyperplasia, reproductive system associateddisorders such as benign prostatic hyperplasia and ovarian cysts,pulmonary fibrosis, endometriosis, fibromatosis, harmatomas,lymphangiomatosis, sarcoidosis, desmoid tumors and the like.

Smooth muscle cell proliferation includes proliferative vasculardisorders, for example, intimal smooth muscle cell hyperplasia,restenosis and vascular occlusion, particularly stenosis followingbiologically- or mechanically-mediated vascular injury, e.g., vascularinjury associated with balloon angioplasty or vascular stenosis.Moreover, intimal smooth muscle cell hyperplasia can include hyperplasiain smooth muscle other than the vasculature, e.g., hyperplasia in bileduct blockage, in bronchial airways of the lung in asthma patients, inthe kidneys of patients with renal interstitial fibrosis, and the like.

Non-cancerous proliferative disorders also include hyperproliferation ofcells in the skin such as psoriasis and its varied clinical forms,Reiter's syndrome, pityriasis rubra pilaris, and hyperproliferativevariants of disorders of keratinization (e.g., actinic keratosis, senilekeratosis), scleroderma, and the like.

Drugs that can be used in combination with the compounds of theinvention to treat a patient with a proliferative disorder such ascancer, or to reduce the likelihood of the reoccurrence of aproliferative disorder such as cancer, include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyturacil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma, antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glyeinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron: azatoxin; azatyrosinc; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoxiraine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamidc-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzclesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline saffonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogs; clotrimazole; collismycinA; collismycin B; conibretastatin A4; combretastatin analogs; eonagenin;crambescidin 816; crisnatol; cryptophycin 8; cryptaphycin A derivatives;curacin A; cyclopentanthraquinones; cycloplatam; cypcmycin; cvtarabineocfosfate; cytolytic factor; cytostatin, dachximab; decitabine;dehydrodidermain B; deslorelin; dexamethasone; dexifosfamide;dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;diethylnorspermine; dihydro-5-azacytidine; 9-dioxamycin; diphenylspirornastine; docosanol; dolasetron; doxifluridine; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur, epirubicin; epristeride;estramustine analogs; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; tilgrastim; finasteride; flavopiridol; tlezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;immunostimulant peptides; insulin-like growth factor-1 receptorinhibitor; interferon agonists; interferons; interleukins; iobenguane;iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin;levamisole; liarozole; linear polyamine analog: lipophilic disaccharidepeptide; lipophilic platinum compounds; lissoelinamide 7; lobaplatin;lombricine; lometrexol; lonidamine; losoxantrone; lovastatin;loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lyticpeptides; maitansine; mannostatin A; marimastat; masoprocol; maspin;matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril;merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor;mifepristone; miltefosine; mirimostim; mismatched double stranded RNA;mitoguizone; mitolactol; mitomycin analogs; mitonafide; mitotoxinfibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; monoclonal antibody, human chorionic gonadotrophin,monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multipledrug resistance gene inhibitor; multiple tumor suppressor 1-basedtherapy; mustard anticancer agent; mycaperoxide B; mycobacterial cellwall extract; myriaporone; N-acetyldinaline; N-substituted benzamides;nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin;nartograstim; medaplatin; nemorubicin; neridronic acid; neutralendopeptidase, nilutamide; nisamycin; nitric oxide modulators; nitroxideantioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone;oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oralcytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin;palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfatesodium; pentostatin; pentrozole; perflubron; perfosfamide; perillylalcohol; phenazinomycin; phenylacetate; phosphatase inhibitors;picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetinA; placetin B; plasminogen activator inhibitor; platinum complex;platinum compounds; platinum-triamine complex; porfimer sodium;portiromycin; prednisone; propyl bis-acridone; prostaglandin J2;proteasome inhibitors; protein A-based immune modulator; protein kinaseC inhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethyleneconjugate; rat antagonists; raltitrexed: ramosetron; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;roquinimex; rubiginone B1; ruboxyl; satingol; saintopin; SarCNU;sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescencederived inhibitor 1; sense oligonucleotides; signal transductioninhibitors; signal transduction modulators; single chain antigen-bindingprotein; sizotiran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustinc; splcnopentin; spongistatin1; squalarnine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide;tauromustine; tazarotene; tccogalan sodium; tegafur; tellurapyrylium;telomerase inhibitors; temoporfin; temozolomide; teniposide;tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline;thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietinreceptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyletiopurpurin; tirapazamine; titanocene bichloride; topsentin;toremifene; totipotent stem cell factor; translation inhibitors;tretinoin; triacetyturidine; triciribine; trimetrexate; triptorelin;tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBCinhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor;urokinase receptor antagonists; vapreotide; variolin B; vector system,erythrocyte gene therapy; velarcsol; veramine; verdins; verteportin;yinorelbine; vinxaltinc; vitaxin; vorozole; zanotcrone; zeniplatin;zilascorb; and zinostatin stirnalamer. Preferred additional anti-cancerdrugs are 5-fluorouracil and leueovorin.

Examples of therapeutic antibodies that can be used in combination withthe compounds of the invention to treat a proliferative disorder such ascancer, or to reduce the likelihood of the reoccurrence of aproliferative disorder such as cancer, include but are not limited toHERCEPTIN® (Trastuzumab) (Genentech, Calif.) which is a humanizedanti-HER2 monoclonal antibody for the treatment of patients withmetastatic breast cancer; REOPRO® (abciximab) (Centocor) which is ananti-glycoprotein IIb/IIIa receptor on the platelets for the preventionof clot formation; ZENAPAX® (dadizutnah) (Roche Pharmaceuticals,Switzerland) which is an immunosuppressive, humanized anti-CD25monoclonal antibody for the prevention of acute renal allograftrejection; PANOREX™ which is a murine anti-17-IA cell surface antigenIgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murineanti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225which is a chimeric anti-EGER IgG antibody (ImClone System); VITAXIN™which is a humanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti CD52IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimericanti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku);LYMPHOCIDE™ which is humanized anti-CD22 IgG antibody (Immunomedics);LYMPHOCIDE™ Y-90 (Immunomedics); Lymphoscan (Tc-99m-labeled;radioimaging; Immunomedics); Nuvion (against CD3; Protein Design Labs);CM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is aprimaticd anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ is aradiolabelled murine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 isa humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatizedanti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (ProteinDesign Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody(Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CAT/BASF);CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is aprimatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham);MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Geninab);CD20-sreptdavidin (+biotin-yttrium 90; NeoRx); CDP571 is a humanizedanti-TNF-αIgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); and CAT-152 is a human anti-TGE-β2 antibody (Cambridge Ab Tech).

Chemotherapeutic agents that can be used in combination with thecompounds of the invention to treat a patient with a proliferativedisorder such as cancer, or to reduce the likelihood of the reoccurrenceof a proliferative disorder such as cancer, include but are not limitedto alkylating agents, antimetabolites, natural products, or hormones.

Examples of alkylating agents useful for the treatment or prevention(reduction in the likelihood of developing or the likelihood ofreoccurrence) of a proliferative disorder such as cancer in the methodsand compositions or the invention include but are not limited to,nitrogen mustards (e.g., mechloroethamine, cyclophosphamide,chlorambucil, melphalan, etc.), ethylenimine and methylmclamineshexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan),nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin,etc,), or triazenes (decarbazine, etc.). Examples of antimetabolitesuseful for the treatment or prevention (reduction in the likelihood ofdeveloping or the likelihood of reoccurrence) of cancer in the methodsand compositions of the invention include but are not limited to folicacid analog (e.g., methotrexate), or pyrimidine analogs (e.g.,fluorouracil, floxouridine, Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin). Examples of natural productsuseful for the treatment or prevention (reduction in the likelihood ofdeveloping or the likelihood of reoccurrence) of cancer in the methodsand compositions of the invention include but are not limited to vincaalkaloids e,g., vinblastin, vincristine), epipodophyllotoxins(e.g.,etoposide, teniposide), antibiotics (e.g., actinomycin D, daunorubicin,doxorubicin, bleomycin, plicamycin, mitomycin), enzymes e.g.,L-asparaginase), or biological response modifiers e.g., interferonalpha). Examples of hormones and antagonists useful for the treatment orprevention (reduction in the likelihood of developing or the likelihoodof reoccurrence) of cancer in the methods and compositions of theinvention include but are not limited to adrenocorticosteroids (e.g.,prednisone), progestins (e,g., hydroxyprogesterone caproate, mcgestrolacetate, medroxyprogesterone acetate), estrogens (e.g.,dicthlystilbestrol, ethinyl estradiol), antiestrogen tamoxifen),androgens testosterone propionate, fluoxymesterone), antiandrogen (e.g.,flutamide), gonadotropin releasing hormone analog (e.g., leuprolide).Other agents that can be used in the methods and with the compositionsof the invention for the treatment or prevention (reduction in thelikelihood of developing or the likelihood of reoccurrence) of cancerinclude platinum coordination complexes (e.g., cisplatin, carboplatin),anthracenedione (e.g., mitoxantrone), substituted urea (e.g.,hydroxyurea), methyl hydrazine derivative (e.g., procarbazine),adrenocortical suppressant (e.g., mitotane, aminoglutethimide).

In one embodiment, the compounds of the invention can be used incombination with an immunotherapeutic agent for the treatment of aproliferative disorder such as cancer, or to reduce the likelihood ofthe reoccurrence of a proliferative disorder such as cancer.Immunotherapy (also called biological response modifier therapy,biologic therapy, biotherapy, immune therapy, or biological therapy) istreatment that uses parts of the immune system to tight disease.Immunotherapy can help the immune system recognize cancer cells, orenhance a response against cancer cells. Immunotherapies include activeand passive immunotherapies. Active immunotherapies stimulate the body'sown immune system while passive immunotherapies generally use immunesystem components created outside of the body.

Examples of active immunotherapies include: cancer vaccines, tumor cellvaccines (autologous or allogeneic), dendritic cell vaccines, antigenvaccines, anti-idiotype vaccines, DNA vaccines, Lymphokine-ActivatedKiller (LAK) Cell Therapy, or Tumor-Infiltrating Lymphocyte (TIL)Vaccine with Interleukin-2 IL-2). Active immunotherapies are currentlybeing used to treat or being tested to treat various types of cancers,including melanoma, kidney (renal) cancer, bladder cancer, prostatecancer, ovarian cancer, breast cancer, colorectal cancer, lung cancer,leukemia, prostate cancer, non-Hodgkin's lymphoma, pancreatic cancer,lymphoma, multiple myeloma, head and neck cancer, liver cancer,malignant brain tumors, and advanced melanoma.

Examples of passive immunothcrapies include: monoclonal antibodies andtargeted therapies containing toxins. Monoclonal antibodies includenaked antibodies and conjugated antibodies (also called tagged, labeled,or loaded antibodies). Naked monoclonal antibodies do not have a drug orradioactive material attached whereas conjugated monoclonal antibodiesare joined to a chemotherapy drug (chemolabeled), a radioactive particle(radiolabeled), or a toxin (immunotoxin). A number of naked monoclonalantibody drugs have been approved for treating cancer, including:

Rituximab (Rituxan), an antibody against the CD20 antigen used to treatB cell non-Hodgkin lymphoma; Trastuzumab (Herceptin), an antibodyagainst the HER2 protein used to treat advanced breast cancer;Alemtuzumab (Campath), un antibody against the CD52 antigen used totreat B cell chronic lymphocytic leukemia (B-CLL), Cetuxirnab (Erbitux),an antibody against the EGFR protein used in combination with irinotecanto treat advanced colorectal cancer and to treat head and neck cancers;and Bevacizumab (Avastin) which is an antiangiogenesis therapy thatworks against the VEGF protein and is used in combination withchemotherapy to treat metastatic colorectal cancer. A number ofconjugated monoclonal antibodies have been approved for treating cancer,including: Radiolabeled antibody Ibritumomab tiuxetan (Zevalin) whichdelivers radioactivity directly to cancerous B lymphocytes and is usedto treat B cell non-Hodgkin lymphoma; radiolabeled antibody Tositumomab(Bexxar) which is used to treat certain types of non-Hodgkin lymphoma;and immunotoxin Gemtuzumab ozogamicin (Mylotarg) which containscalieheamicin and is used to treat acute myelogenous leukemia (AML).BL22 is a conjugated monoclonal antibody currently in testing fortreating hairy cell leukemia and there are several immunotoxin clinicaltrials in progress for treating leukemias, lymphomas, and brain tumors.There are also approved radiolabeled antibodies used to detect cancer,including OncoSeint for detecting colorectal and ovarian cancers andProstaScint for detecting prostate cancers. Targeted therapiescontaining toxins are toxins linked to growth factors and do not containantibodies. An example of an approved targeted therapy containing toxinsis denileukin diftitox (Ontak) which is used to treat a type of skinlymphoma (cutaneous T cell lymphoma).

Examples of adjuvant immunotherapies include: cytokines, such asgranulocyte-macrophage colony-stimulating factor (GM-CSF),granulocyte-colony stimulating factor (G-CSF), macrophage inflammatoryprotein (MIP)-1-alpha, interleukins (including IL-1, IL-2, IL-4, IL-6,IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27), tumor necrosis factors(including TNF-alpha), and interferons (including IFN-alpha, IFN-beta,and IFN-gamma); aluminum hydroxide (alum); Bacille Calmette-Guérin(BCG); Keyhole limpet hemocyanin (KLH); Incomplete Freund's adjuvant(IFA); QS-21; DETOX; Levamisole; and Dinilrophenyl (DNP). Clinicalstudies have shown that combining IL-2 with other cytokines, such asIFN-alpha, can lead to a synergistic response.

Several types of immunotherapies are being used to treat melanomapatients. IFN-alpha and IL-2 are approved for treatment of people withmetastatic melanoma. BCG is being tested in combination with melanomavaccines and other immuitothcrapies. Tumor-infiltrating lymphocytes havebeen shown to shrink melanoma tumors in a phase 1 clinical trial. Humanmonoclonal antibodies to ganglioside antigens have been shown to regresscutaneous recurrent melanoma tumors. Some autologous and allogeneictumor cell vaccines, antigen vaccines (including polyvalent antigenvaccines), viral vaccines, and dendritic cell vaccines have also beenshown to shrink tumors. Clinical trials continue for these and othermelanoma immunotherapies. Melanoma patients with a high IgM responseoften survive better than those who elicit no or low IgM antibodies(Morton et al., 1992). Combined IL-12/TNF-alpha immunotherapy has beenshown to significantly retard tumor growth in three tumor models in mice(B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma) ascompared with controls and mice treated with either cytokine alone.IFN-alpha is approved for the treatment of malignant melanoma, chronicrnyelogenous leukemia (CML), hairy cell leukemia, and Kaposi's sarcoma.

Several types of immunotherapies are being used to treat patients thathave renal cancer. IFN-alpha and IL-2 are approved for treatment ofpeople with metastatic renal (kidney) cancer. A combination therapyusing IL-2, interferon, and chemotherapy is being tested for treatmentof renal cancer. Treatment with a tumor cell vaccine plus the adjuvantBCG has been shown to shrink tumors in some advanced renal cancerpatients. DNA vaccines and tumor-infiltrating lymphocytes are also beingtested as treatments for renal cancer. Chimeric bispecific G250/anti-CD3monoclonal antibodies have been shown to mediate cell lysis of renalcell carcinoma cell lines by cloned human CD8+T cells or by IL2stimulated peripheral blood lymphocytes.

As used herein, a “microtubulin stabilizer” means an anti-cancer agentwhich acts by arresting cells in the G2-M phases due to stabilization ofmicrotubules. Agents which are microtubulin stabilizers can be used incombination with the compounds or the invention to treat patients havinga proliferative disorder such as cancer, or to reduce the likelihood ofthe reoccurrence of a proliferative disorder such as cancer. Examples ofmicrotubulin stabilizers include taxol and taxol analogs. Additionalexamples of microtubulin stabilizers included without limitation thefollowing marketed drugs and drugs in development: Discodermolide (alsoknown as NVP-XX-A-296); Epothilones (such as Epothilone A, Epothilone B,Epothilone C (also known as desoxyepothilone A or dEpoA); Fpothilonc D(also referred to as KOS-862, dEpoB, and desoxyepothilone B); EpothiloneE; Epothilone F; Epothilone B N-oxide; Epothilone A N-oxide;16-aza-epothilone B; 21-aminoepothilone B (also known BMS-310705);21-hydroxyepothilone D (also known as Desoxyepothilone F and dEpoF),26-fluoroepothilone); FR-182877 (Fujisawa, also known as WS-9885B),BSF-223651 (BASF, also known as ILX-651 and LU-223651); AC-7739(Ajinomoto, also known as AVE-8063A and CS-39.HCl); AC-7700 (Ajinomoto,also known as AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A);Fijianolide B; Laulimalide; Caribaeoside; Caribaeolin; Taccalonolide;Eleutherobin; Sarcodictyin; Laulimalide; Dictyostatin-1; Jatrophaneesters; and analogs and derivatives thereof.

As used herein, a “microtubulin inhibitor” means an anti-cancer agentwhich acts by inhibiting tubulin polymerization or microtubule assembly.Agents which are microtubulin inhibitors can be used in combination withthe compounds of the invention to treat patients having a proliferativedisorder such as cancer, or to reduce the likelihood the reoccurrence ofa proliferative disorder such as cancer. Examples of microtubulininhibitors include without limitation the following marketed drugs anddrugs in development; Erbulozole (also known as R-55104); Dolastatin 10(also known as DLS-10 and NSC-376128); Mivobulin isethionate (also knownas CI-980); Vincristinc; NSC-639829, ABT-751 (Abbot, also known asE-7010); Altorhyrtins (such as Altorhyrtin A and Altorhyrtin C);Spongistatins (such as Spongistatin 1, Spongistatin 2, Spongistatin 3,Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7,Spongistatin 8, and Spongistatin 9); Cemadotin hydrochloride (also knownas LU-103793 and NSC-D-669356); Auristatin PE (also known asNSC-654663); Soblidotin (also known as TZT-1027), LS-4559-P (Pharmacia,also known as LS-4577); LS-4578 (Pharmacia, also known as LS-477-P);LS-4477 (Pharmacia), LS-4559 (Pharmacia); RPR-112378 (Aventis);Vincristine sulfate; DZ-3358 (Daiichi); GS-164 (Takeda); GS-I98(Takeda); KAR-2 (Hungarian Academy of Sciences); SAH-49960(Lilly/Novartis); SDZ-268970 (Lilly/Novartis); AM-97 (Armad/KyowaHakko); AM-132 (Armad); AM-138 (Armad/Kyowa Hakko); IDN-5005 (Indenal;Cryptophycin 52 (also known as LY-355703); Vitilevuamide; Tubulysin A,Canadensol, Centaureidin (also known as NSC-106969); T-138067 (Tularik,also known as T-67, TL-138067 and TI-138067); COBRA-1 (Parker HughesInstitute, also known as DDE-261 and WHI-261); H10 (Kansas StateUniversity); H16 (Kansas State University); Oncocidin A1 (also known asBTO-956 and DIME); DDE-313 (Parker Hughes Institute); SPA -2 (ParkerHughes institute); SPA-1 (Parker Ilughes Institute, also known asSPIKET-P); 3-IAABU (Cytoskeleton/Mt. Sinai School of Medicine, alsoknown as MF-569); Narcosine (also known as NSC-5366); Nascapine, D-2485I(Asta Medica), A-105972 (Abbott); Hemiasterlin; 3-BAABU(Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-191); TMPN(Arizona State University); Vanadocene acetylacetonate; T-138026(Tularik); Monsatrol; Inanocine (also known as NSC-698666), 3-IAABE(Cytoskeleton/Mt. Sinai School of Medicine); A-204197 (Abbott); T-607(Tularik, also known as T-900607), RPR-115781 (Aventis); Eleutherobins(such as Desmethyleleutherobin, Desaetyleleutherobin, Isoeleutherobin A,and Z-Eleutherobin); Halichondrin B; D-64131 (Asta Medica); D-68144(Asta Medica); Diazonamide A; A-293620 (Abbott); NPI-2350 (Nereus);TUB-245 (Aventis); A-259754 (Abbott); Diozostatin; (−)-Phenylahistin(also known as NSCL-96F037); D-68838 (Asta Medica); D-68836 (AstaMedica); Myoseverin B; D-43411 (Zentaris, also known as D-81862);A-289099 (Abbott); A-318315 (Abbott); HTI-286 (also known as SPA-110,tritluoroacetate salt) (Wyeth); D-82317 (Zentaris); D-82318 (Zentaris);SC-12983 (NCI); Resverastatin phosphate sodium; BPR-0Y-007 (NationalHealth Research Institutes); SSR-250411 (Sanofi); Combretastatin A4; andanalogs and derivatives thereof.

Paclitaxel also referred to as “Taxol®”, is a well-known anti-cancerdrug which acts by enhancing and stabilizing microtubule formation. Thestructure of paclitaxel is shown in FIG. 1. Many analogs of paclitaxelare known, including docetaxol, the structure of which is shown in FIG.2. Docetaxol is also referred to ua “Taxotere®”. The structures of otherpaclitaxel analogs are shown in FIGS. 3-23. These compounds have thebasic taxane skeleton as a common structure feature and have also beenshown to have the ability to arrest cells in the G2-M phases due tostabilization of microtubules. Thus, it is apparent from FIGS. 3-23 thata wide variety of substituents can decorate the taxane skeleton withoutadversely affecting biological activity. It is also apparent that zero,one or both of the cyclohexane rings of a paclitaxel analog can have adouble bond at the indicated positions. For clarity purposes, the basictaxane skeleton is shown below in Structural Formula (XXVI):

Double bonds have been omitted from the cyclohexane rings in the taxaneskeleton represented by Structural Formula (XXVI). The basic taxaneskeleton can include zero or one double bond in one or both cyclohexanerings, as indicated in FIGS. 3-23 and Structural Formulas (XXVII) and(XXVIII) below. A number of atoms have also been omitted from StructuralFormula (XXVI) to indicate sites in which structural variation commonlyoccurs among paclitaxel analogs. For example, substitution on the taxaneskeleton with simply an oxygen atom indicates that hydroxyl, acyl,alkoxy or another oxygen-bearing substituent is commonly found at thesite. These and other substitutions on the taxane skeleton can be madewithout losing the ability to enhance and stabilize microtubuleformation. Thus, the term “paclitaxel analog” is defined herein to meana compound which has the basic taxane skeleton and which promotesmicrotubule formation. Paclitaxel analogs may be formulated as ananoparticle colloidal composition to improve the infusion time and toeliminate the need to deliver the drug with Cremophor which causeshypersensitivity reactions in some patients. An example of a paelitaxelanalog formulated as a nanopartiele colloidal composition is Abraxanewhich is a nanoparticle colloidal composition of protein-stabilizedpaclitaxel that is reconstituted in saline.

Typically, the paelitaxel analogs used herein are represented byStructural Formula (XXVII) or (XXVIII):

R₁₀ is a lower alkyl group, a substituted lower alkyl group, a phenylgroup, a substituted phenyl group, —SR₁₉, —NHR₁₉ or —OR₁₉.

R₁₁ is a lower alkyl group, a substituted lower alkyl group, an arylgroup or a substituted aryl group,

R₁₂ is —H, —OH, lower alkyl, substituted lower alkyl, lower alkoxy,substituted lower alkoxy, —O—C(O)-(lower alkyl), —O—C(O)-(substitutedlower alkyl), —O—CH₂—O-(lower alkyl) —S—CH₂—O—-(lower alkyl).

R₁₃ is —H, —CH₃, or, taken together with R₁₄, —CH₂—.

R₁₄ is —H, —OH, lower alkoxy, —O—C(O)-(lower alkyl), substituted loweralkoxy, —O—C(O)-(substituted lower alkyl), —O—CH₂—O—P(O)(OH)₂,—O—CH₂—O-(lower alkyl), —O—CH₂—S-(lower alkyl) or, taken together withR₂₀, a double bond.

R₁₅—H, lower acyl, lower alkyl, substituted lower alkyl, alkoxymethyl,alkthiomethyl, —OC(O)—O(lower alkyl), —OC(O)—O(substituted lower alkyl),—OC(O)-NH(lower alkyl) or —OC(O)—NH(substituted lower alkyl).

R₁₆ is phenyl or substituted phenyl.

R₁₇ is —H, lower acyl, substituted lower acyl, lower alkyl, substituted,lower alkyl, (lower alkoxy)methyl or (lower alkyl)thiomethyl.

R₁₈—H, —CH₃ or, taken together with R₁₇ and the carbon atoms to whichR₁₇ and R₁₈ are bonded, a five or six membered a non-aromaticheterocyclic ring.

R₁₉ is a lower alkyl group, a substituted lower alkyl group, a phenylgroup, a substituted phenyl group.

R₂₀ is —H or a halogen.

R₂₁ is —H, lower alkyl, substituted lower alkyl, lower acyl orsubstituted lower acyl.

Preferably, the variables in Structural Formulas (XXVII) and (XXVIII)are defined as follows: R₁₀ is phenyl, tert-butoxy, —S—CH₂—CH—(CH₃)₂,—S—CH(CH₃)₃, —S—(CH₂)₃CH₃, —O—CH((CH₃)₃, —NH—CH(CH₃)₃, —CH═C(CH₃)₂ orpara-chlorophenyl; is phenyl, (CH₃)₂CHCH₂—, -2-furanyl, cyclopropyl orpara-toluyl; R₁₂ is —H, —OH, CH₃CO— or —(CH₂)₂-N-morpholino; R₁₃ ismethyl, or, R₁₃ and R₁₄, taken together, are —CH₂—;

R₁₄ is —H, —CH₂SCH₃ or —CH₂—O—P(O)(OH)₂; R₁₅ is CH₃CO—;

R₁₆ is phenyl; R₁₇—H, or, R₁₇ and R₁₈, taken together, are —O—CO—O—;

R₁₈ is —H; R₂₀ is —H or —F; and R₂₁ is —H, —C(O)—CHBr—(CH₂)₁₃—CH₃ or—C(O)—(CH₂)₁₄—CH₃; —C(O)—CH₂—CH(OH)—COOH,—C(O)—CH₂—O—C(O)—CH₂CH(NH₂)—CONH₂, —C(O)—CH₂—O—CH₂CH₂OCH₃ or—C(O)—O—C(O)—CH₂CH₃.

A paclitaxel analog can also be bonded to or be pendent from apharmaceutically acceptable polymer, such as a polyacrylamide. Oneexample of a polymer of this type is shown in FIG. 24. The term“paclitaxel analog”, as it is used herein, includes such polymers.

In some embodiments, paclitaxel analogs have a taxane skeletonrepresented by Structural Formula XXIX, wherein Z is O, S, or NR.Paclitaxcl analogs that have the taxane skeleton shown in StructuralFormula XXIX can have various substituents attached to the taxaneskeleton and can have a double bond in zero, one or both of thecyclohexane rings as shown, for example in FIGS. 3-23.

Various paclitaxel analogs and paclitaxel formulations are described inHennenfent et al. (2006) Annals of Oncology 17:735-749; Gradishar (2006)Expert Opin. Pharmacother. 7(8):1041-53; Attard et al. (2006) PatholBiol 54(21:72-84; Straubinger et al. (2005) Methods Enzymol. 391:97-117;Ten Tije et al. (2003) Clin Pharmacokinet. 42(7):665-85; and Nuijen etal. (2001) Invest New Drugs. 19(21:143-53, the entire teachings of whichare incorporated herein by reference.

An “alkyl group” is saturated straight or branched chain linear orcyclic hydrocarbon group. Typically, a straight chained or branchedalkyl group has from 1 to about 20 carbon atoms, preferably from 1 toabout 10, and a cyclic alkyl group has from 3 to about 10 carbon atoms,preferably from 3 to about 8. An alkyl group is preferably a straightchained or branched alkyl group, e.g., methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl oroctyl, or a cycloalkyl group with 3 to about 8 carbon atoms. A C₁-C₈straight chained or branched alkyl group or a C₃-C₈ cyclic alkyl groupis also referred to as a “lower alkyl” group. Suitable substituents foran alkyl group are those which do not substantially interfere with theanti-cancer activity of the disclosed compounds. Suitable substituentare as described below for aliphatic groups. Preferred substituents onalkyl groups include, —OH, —NH₂, —NO₂, —CN, —COOH, halogen, aryl, C₁₋C₈alkoxy, C₁-C₈ haloalkoxy and —CO(C₁-C₈ alkyl). More preferredsubstituents on alkyl groups include —OH , halogen, phenyl, benzyl,pyridyl, and C₁-C₈ alkoxy. More preferred substituents on alkyl groupsinclude —OH, halogen, and C₁-C₄ alkoxy.

The terms “lower alkoxy” and “lower acyl” mean to —O-(lower alkyl),—C(O)-(lower alkyl), respectively. The terms “substituted lower alkoxy”and “substituted lower acyl” mean —O-(substituted lower alkyl) and—C(O)-(substituted lower alkyl), respectively.

An aliphatic group is a straight chained, branched or cyclicnon-aromatic hydrocarbon which is completely saturated or which containsone or more units of unsaturation. Typically, a straight chained orbranched aliphatic group has from 1 to about 20 carbon atoms, preferablyfrom 1 to about 10, and a cyclic aliphatic group has from 3 to about 10carbon atoms, preferably from 3 to about 8. An aliphatic group ispreferably a straight chained or branched alkyl group, e.g., methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, Leo-butyl, pentyl,hexyl, pentyl or octyl, or a cycloalkyl group with 3 to about 8 carbonatoms. A C₁-C₈ straight chained or branched alkyl group or a C₃-C₈cyclic alkyl group is also referred to as a “lower alkyl” group.

The term “aromatic group” may be used interchangeably with “aryl,” “arylring,” “aromatic ring,” “aryl group” and “aromatic group.” Aromaticgroups include carbocyclic aromatic groups such as phenyl, naphthyl, andanthracyl, and hetcroaryl groups such as imidazolyl, thienyl, furanyl,pyridyl, pyrimidyl, pyranyl, pyravolyl, pyrroyl, pyrazinyl, thiazolc,oxazolyl, and tetrazole. The term “heteroaryl group” may be usedinterchangeably with “heteroaryl,” “heteroaryl ring,” “heteroaroinaticring” and “heteroaromatic group,” fleteroaryl groups are aromatic groupsthat comprise one or more heteroatom, such as sulfur, oxygen andnitrogen, in the ring structure. Preferably, heteroaryl groups comprisefrom one to four heteroatoms.

Aromatic groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic aromatic ring or heteroaryl ring is used to one ormore other heteroaryl rings. Examples include benzothienyl,benzofuranyl, indolyl, quinolinyl, benzothiazole, benzooxazole,benzimidazole, quinolinyl, isoquinolinyl and isoindolyl.

Non-aromatic heterocyclic rings are non-aromatic rings which include oneor more heteroatoms such as nitrogen, oxygen or sulfur in the ring. Thering can he five, six, seven or eight-membered. Preferably, heterocyclicgroups comprise from one to about four heteroatoms. Examples includetetrahydrofuranyl, tetrithydrothiophenyl, morphohno, thiomorpholino,pyrrolidinyl, piperazinyl, piporidinyl, and thiazolidinyl.

Examples of suitable substituents include —R^(a), —OH, —Br, —Cl, —I, —F,—OR^(a), —O—COR^(a), −COR^(a), —CN, —NO₂, —COOH, —SO₃H, —NH₂, —NHR^(a),—N(R^(a)R^(b)), —COOR^(a), —CHO, —CONH₂, —CONHR^(a), —CON(R^(a)R^(b)),—NHCOR^(a), —NR^(c)COR^(a), —NHCONH₂, —NHCONR^(a)H, —NHCON(R^(a)R^(b)),—NR^(c)CONH₂, —NR^(c)CONR^(a)H, —NR^(c)CON(R^(a)R^(b)), —C(═NH)—NH₂m—C(═NH)—NHR^(a), —C(═NH)—N(R^(a)R^(b)), —C(═NR^(c))—NH₂,—C(═NR^(c))—NH₂, —C(═NR^(c))—NHR^(a)m —C(═NR^(c))—N(R^(a)R^(b)),—NH—C(—NH)—NH₂, —NH—C(═NH)—NHR^(a), —NH—C(═NH)—N(R^(a)R^(b)),—NH—C(═NR^(c))—NH₂, —NH—C(═NR^(c))—NHR^(a)m—NH—C(═NR^(c))—N(R^(a)R^(b)), —NR^(d)H—C(═NH)—NH₂,—NR^(d)—C(═NH)—NHR^(a), —NR^(d)—C(═NH)—N(R^(a)R^(b)),—NR^(d)—C(═NR^(c))—NH₂, —NR^(d)—C(═NR^(c))—NHR^(a),—NR^(d)—C(═NR^(c))—N(R^(a)R^(b)), —NHNH₂, —NHNHR^(a), —NHR^(a)R^(b),—SO₂NH₂, —SO₂NHR^(a), —SO₂NR^(a)R^(b), —CH═CHR^(a), —CH═CR^(a)R^(b),—CR^(c)═CR^(a)R^(b), —CR^(c)═CHR^(a), —CR^(c)═CR^(a)R^(b), —CCR^(a),—SH, —SR^(a), —S(O)R^(a), —S(O)₂R^(a).

R^(a)-R^(d) are each independently an alkyl group, aromatic group,non-aromatic heterocyclic group or —N(R^(a)R^(b)), taken together, forina non-aromatic heterocyclic group.

In co-therapy in combination with one or more other therapeutic agents(e.g., paclitaxel or paclitaxel analogs), the compound or thepharmaceutical composition disclosed herein can be administeredsimultaneously or separately with the other therapeutic agent(s). Theexact details of the administration will depend on the pharmacokineticsof the two substances in the presence of each other, and can includeadministering two substances substantially at the same time, and onesubstance within a certain time period te.g., within 24 hours) ofadministration of the other, if the pharmacokinetics are suitable.Designs of suitable dosing regimens are routine for one skilled in theart. In particular embodiments, two substances will be administeredsubstantially simultaneously, i.e. within minutes of each other, or in asingle composition that comprises both substances.

In one embodiment, the present invention is directed to a methodtreating a subject with a Hsp70-responsive disorder. The methodcomprises administering to the subject an effective amount of a compoundor a pharmaceutical composition describe herein.

As used herein, “Hsp70” includes each member of the family of heat shockproteins having a mass of about 70-kiloDaltons, including forms such asconstituitive, cognate, cell-specific, glucose-regulated, inducible,etc. Examples of specific Hsp70 proteins include hsp70, hsp70hom; hsc70;Grp78/BiP: mt-hsp70/Grp75, and the like). Typically, the disclosedmethods increase expression of inducible Hsp70. Functionally, the 70-kDaHSP (HSP70) family is a group of chaperones that assist in the folding,transport, and assembly of proteins in the cytoplasm, mitochondria, andendoplasmic reticulum. In humans, the Hsp70 family encompasses at least11 genes encoding a group of highly related proteins. See, for example,Tavaria, et al., Cell Stress Chaperones, 1996; 1(1):23-28; Todryk, etal., Immunology, 2003, 110(1): 1-9; and Georgopoulos and Welch, Annu RevCell Biol. 1993; 9:601- 634; the entire teachings of these documents areincorporated herein by reference.

As used herein, an “Hsp70-responsive disorder” is a medical conditionwherein stressed cells can he treated by increased Hsp70 expression.Such disorders can be caused by a wide variety of cellular stressors,including, but not limited to Alzheimer's disease; Huntington's disease;Parkinson's disease; spinal/bulbar muscular atrophy (e.g., Kennedy'sdisease), spinocerebellar ataxic disorders, and other neuromuscularatrophies; familial amyotrophic lateral sclerosis; ischemia; seizure;hypothermia; hyperthermia; burn trauma; atherosclerosis; radiationexposure; glaucoma; toxin exposure; mechanical injury; inflammation;autoimmune disease; infection (bacterial, viral fungal, or parasitic);and the like.

In some embodiments, the Hsp70-responsive disorder is aneurodegenerative disorder. As used herein, a neurodegenerative disorderinvolves degradation of neurons such as cerebral, spinal, and peripheralneurons (e.g., at neuromuscular junctions), more typically degradationof cerebral and spinal neurons, or in preferred embodiments, degradationof cerebral neurons. Neurodegenerative disorders can include Alzheimer'sdisease; Huntington's disease; Parkinson's disease; spinal/bulbarmuscular atrophy and other neuromuscular atrophies; and familialamyotrophic lateral sclerosis or other diseases associated withsuperoxide dismutase (SOD) mutations. Neurodegencrative disorders canalso include degradation of neurons caused by ischemia, seizure, thermalstress, radiation, toxin exposure, infection, injury, and the like.

In some embodiments, the Hsp70-responsive disorder is a disorder ofprotein aggregation/misfolding, such as Alzheimer's disease;Huntington's disease; Parkinson's disease; spongiform encephalopathies;and the like.

In another embodiment the Hsp70-responsive disorder is a treatment orcondition which causes or may cause nerve damage. The compounds for usein the methods of the present invention can be used to reduce or prevent(inhibit the onset of) nerve damage (i.e., provide neuroprotcction) in asubject i) suffering from a condition which causes or may cause nervedamage or ii) receiving treatment which causes or may cause nervedamage. In one aspect, the treatment which causes or may cause nervedamage is radiation therapy. In another aspect, the treatment ischemotherapy. In one aspect, the chemotherapy comprises administering anantimitotic agent (e.g. vincristine, vinorelbine, paclitaxel, or apaclitaxel analog). In one aspect, the chemotherapy comprisesadministering paclitaxel. In another aspect, the chemotherapy comprisesadministering a platinum derivative (e.g. cisplatin, earboplatin, oroxahplatin). In certain embodiments, the compounds for use in themethods of the present invention can be administered simultaneously as acombination therapy with the treatment which causes or may cause nervedamage. In other embodiments the compounds for use in the methods of thepresent invention can be administered before or after the treatmentwhich causes may cause nerve damage. In certain embodiments thecompounds for use in the methods of the present invention can beadministered between 30 minutes and 12 hours, between 1 hour and 6before or after the treatment which causes or may cause nerve damage.

Nerve damage may be caused by a number of treatments including, but notlimited to, radiation therapy; chemotherapy, e.g. cisplatin,carboplatin, oxaliplatin, vincristine, vinblastine, vinorelbine,vindesine, ifosfamide, methotrexate, cladribine, altretamine,fludarabine, procarbazine, thiotepa, teniposide, arsenic trioxide,alemtuzumab, capecitabine, dacarbazine, denileukin diftitox, interferonalpha, liposomal daunorubicin, tretinoin, etoposide/VP-16, cytarabine,hexamethylmelamine, suramin, paclitaxcl, docetaxel, gemcitibine,thalidomide, and bortezomib; heart or blood pressure medications, e.g.amiodarone, hydralazine, digoxin, and perhxiline; medications to fightinfection, e.g. metronidazole, nitrofurantoin, thalidomide, and INH;medications to treat skin conditions, e.g. dapsone; anticonvulsants,e.g. phenytoin; anti-alcohol medications, e.g. disulfiram; HIVmedications, e.g. zidovudine, didanonsine, stavudine, zalcitabine,ritonavir, d4T, ddC, ddl, and amprenavir, cholesterol medications, e.g.lovastatin, pravastatin, indapamid, simvastatin, fluvastatin,atorvastatin, cerivastatin, and gemfibrozil: antirheumatics, e.g.chloroquine, cholchicine, organic gold, and penicillamine: nitrousoxide; lithium; and ergots.

In some embodiments, the Hsp70-responsive disorder is ischemia. Ischemiacan damage tissue through multiple routes, including oxygen depletion,glucose depletion, oxidative stress upon reperfusion, and/or glutamatetoxicity, and the like. Ischemia can result from an endogenous condition(e.g., stroke, heart attack, and the like), from accidental mechanicalinjury, from surgical injury (e.g., reperfusion stress on transplantedorgans), and the like. Alternatively, tissues that can be damaged byischemia include neurons, cardiac muscle, liver tissue, skeletal muscle,kidney tissue, pulmonary tissue, pancreatic tissue, and the like. In onepreferred embodiment, the Hsp70-responsive disorder is cerebral orspinal ischemia. In another preferred embodiment, the Hsp70-responsivedisorder is cardiac ischemia.

In various embodiments, the Hsp70-responsive disorder is seizure, e.g.,epileptic seizure injury-induced seizure, chemically-induced seizure,and the like.

In some embodiments, the Hsp70-responsive disorder is due to thermalstress. Thermal stress includes hyperthermia (e.g., from fever, heatstroke, burns, and the like) and hypothermia. In a preferred embodimentthe disorder is hyperthermia. In another preferred embodiment, theHsp70-responsive disorder is burn trauma.

In preferred embodiments, the Hsp70-responsive disorder isatherosclerosis.

In various embodiments, the Hsp70-responsive disorder is radiationdamage, e.g., due to visible light, ultraviolet light, microwaves,cosmic rays, alpha radiation, beta radiation, gamma radiation, X-rays,and the like. For example, the damage could be radiation damage tonon-cancerous tissue in a subject treated for cancer by radiationtherapy. In a preferred embodiment, the Hsp70-responsive disorder isradiation damage from visible light or ultraviolet light.

In various embodiments, the Hsp70-responsive disorder is mechanicale.g., trauma from surgery, accidents, certain disease conditions (e.g.,pressure damage in glaucoma) and the like. In a preferred embodiment,the Hsp70-responsive disorder is cerebral or spinal trauma. In anotherpreferred embodiment, the Hsp70-responsive disorder is glaucoma (leadingto pressure damage to retinal ganglions).

In various embodiments, the Hsp70-responsive disorder is exposure to atoxin. In preferred embodiments, the Hsp70-responsive disorder isexposure to a neurotoxin selected from methamphetamine, antiretroviralHIV therapeutics (e.g., nucleoside reverse transcriptase inhibitors;heavy metals (e.g., mercury, lead, arsenic, cadmium, compounds thereof,and the like), amino acid analogs, chemical oxidants, ethanol,glutamate, metabolic inhibitors, antibiotics, and the like.

The present invention also provides a method of treating a subject witha natural killer, cell-responsive disorder, wherein the method comprisesadministering to the subject an effective amount of a compound or apharmaceutical composition describe herein.

Certain compounds of the invention also increase Natural Killer (NK)cell activity. As used herein, a “NK cell-responsive disorder” is amedical condition which is improved by an increased in NK cell activity.For example, a subject with a NK cell-responsive disorder may needimmune system augmentation because of infection or the possibilitythereof. In some embodiments, such a subject can have an infection (orhas been exposed to an infectious environment where pathogens arepresent, e.g., in a hospital) the symptoms of which may be alleviated bythe methods disclosed herein. For example, a subject in need oftreatment can have an infection (bacterial, viral, fungal, orparasitical (protozoal) for which the disclosed methods of activating NKcells can be a treatment.

In some embodiments, a subject having an NK cell-responsive disorder hasan immunodeficiency. Such a subject is in need of or can benefit fromprophylactic therapy, for example, a subject that has incomplete,damaged or otherwise compromised defenses against infection, or issubject to an infective environment, or the like. For example, a subjectcan be in an infectious environment where pathogens are present, e.g.,in a hospital; can have an open wound or burn injury; can have aninherited or acquired immune deficiency (e.g., severe combinedimmunodeficiency or “bubble boy” syndrome, variable immunodeficiencysyndrome acquired immune deficiency syndrome (AIDS), or the like); canhave a depressed immune system due to physical condition, age, toxinexposure, drug effect (immunosuppressants, e.g., in a transplantrecipient) or side effect (e.g., due to an anticancer agent); or thelike.

In same embodiments, NK cell activity can be increased in subjects thathave decreased or deficient NK cell activity, in conditions such aschronic fatigue syndrome (chronic fatigue immune dysfunction syndrome)or Epstein-Barr virus infection, post viral fatigue syndrome,post-transplantation syndrome (especially allogeneic transplants) orhost-graft disease, exposure to drugs such as anticancer agents ornitric oxide synthase inhibitors, natural aging, and variousimmunodeficient conditions such as severe combined immunodeficiency,variable immunodeficiency syndrome, and the like.

In some embodiments, a subject having an NK cell-responsive disorder isin need of treatment for bacteremia. Bacteremia is the condition ofbacterial infection in the bloodstream. Septic shock includes seriouslocalized or bacteremic infection accompanied by systemic inflammation,in other words sepsis with hypoperfusion and hypotension refractory tofluid therapy. Sepsis, or systemic inflammatory response syndrome,includes various severe conditions such as infections, pancreatitis,burns, trauma) that can cause acute inflammation. Septic shock istypically related to infections by gram-negative organisms,staphylococci, or meningococci. Septic shock can be characterized byacute circulatory failure, typically with hypotension, and multiorganfailure.

Transient bacteremia can be caused by surgical or trauma wounds.Gram-negative bacteremia can be intermittent and opportunistic; althoughit may have no effect on a healthy person, it may be seriously importantin immunocompromised patients with debilitating underlying diseases,after chemotherapy, and in settings of malnutrition. The infection cantypically be in the lungs, in the genitouritory (GU) or gastrointestinal(GI) tract, or in soft tissues, e.g., skin in patients with decubitusulcer, oral ulcers in patients at risk, and patients with valvular heartdisease, prosthetic heart valves, or other implanted prostheses.

Typically, gram-negative bacteremia can manifest in chronically ill andimmunocompromised patients. Also in such patients, bloodstreaminfections can be caused by aerobic bacilli, anaerobes, and fungi.Bacteroides can lead to abdominal and pelvic infective complications,especially in females. Transient or sustained bacteremia can typicallyresult in metastatic infection of the meninges or serous cavities, suchas the pericardium or larger joints. Enterococcus, staphylococcus, orfungus can lead to endocarditis, but is less common with gram-negativebacteremia. Staphylococcal bacteremia can be typical of IV drug users,and can be a typical cause of gram-positive bacterial endocarditis.

The incidence of systemic fungal infections has undergone a significantincrease, particularly in humans, due in part to increases in the numberof subjects with compromised immune systems, for example, the elderly,AIDS patients, patients undergoing chemotherapy, burn patients, patientswith diabetic ketoacidosis, and transplant patients on immunosuppressivedrugs. A study found that about 40% or deaths from infections acquiredduring hospitalization were due to mycoses; see Sternberg et. al,Science. Vol. 266, (1994), pp. 1632-1634, the entire teachings of whichare incorporated herein by reference.

In various embodiments, the subject having an NK cell-responsivedisorder can be in need of treatment for a fungal infection, such as apathogenic dermatophyte, a pathogenic filamentous fungus, and/or apathogenic non-filamentous fungus, e.g., a yeast, or the like.Pathogenic dermatophytes can include, e.g., species of the generaTrichophyton, Tinea, Microsporum, Epidermophyton, or the like.Pathogenic filamentous fungus can include, e.g., species of genera suchas Aspergillus, Histoplasma, Cryptococcus, Microsporum, or the like.Pathogenic non-filamentous fungus, e.g., yeasts, can include, forexample, species of the genera Candida, Malassezia, Trichosporon,Rhodotorula, Torulopsis, Blastomyces, Paracoccidioides, Coccidioides, orthe like. In various embodiments, the subject can be treated for afungal infection from a species of the genera Aspergillus orTrichophyton. Species of Trichophyton can include, for example,Trichophyton mentagrophytes, Trichophyton rubrum, Trichophytonschoenleinii, Trichophyton tonsurans, Trichophyton verrucosum, andTrichophyton violaceum. Species of Aspergillus can include, for example,Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Aspergillus amstelodami, Aspergillus candidus, Aspergillus carneus,Aspergillus nidulans, A oryzae, Aspergillus restrictus, Aspergillussydowi, Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor,Aspergillus caesiellus, Aspergillus clavatus, Aspergillus avenaceus, andAspergillus deflectus. In some embodiments, the subject can be treatedfor a fungal infection from a pathogenic dermatophyte, e.g.,Trichophyton (e.g., Trichophyton rubrum), Tinea, Microsporum, orEpidermophyton, or Cryptococcus Cryptococcus neoformans) Candida (e.g.,Candida albicans), Paracoccidioides (e.g., Paracoccidioidesbrasiliensis), or Coccidioides (e.g., Coccidioides immitis). Inparticular embodiments, the subject can be treated for a fungalinfection from Trichophyton rubrum, Cryptococcus neoformans, Candidaalbicans, Paracoccidioides brusiliensis, or Coccidioides immitis.

Thus, in various embodiments, a subject can have an infection caused bya fungus selected from the genera Trichophyton, Tinea, Microsporum,Epidermophyton, Aspergillus, Histoplasma, Cryptococcus, Microsporum,Candida, Malassezia, Trichosporon, Rhodotorula, Torulopsis, Blastomyces,Paracoccidioides, and Coccidioides. In some embodiments, the subject canhave an infection caused by a fungus selected from the generaTrichophyton, Tinea, Microsporum, Epidermophyton; Cryptococcus, Candida,Paracoccidioides, and Coccidioides. In certain embodiments, the subjectcan have an infection caused by a fungus selected from Trichophytonrubrum, Cryptococcus neoformans, Candida albicans, Paracoccidioidesbrasiliensis, and Coccidioides immitis.

In various embodiments, the subject having an NK cell-responsivedisorder can be in need of treatment for a bacterial infection caused,for example, by a bacteria of a genus selected from Allochromatium,Acinetobacter, Bacillus, Campylobacter, Chlamydia, Chlamydophila,Clostridium, Citrobacter, Escherichia, Enterobacter, Enterococcus,Francisella, Haemophilus, Helicobacter, Klebsiella, Listeria, Moraxella,Mycobacterium, Micrococcus, Neisseria, Proteus, Pseudomonas, Salmonella,Serratia, Shigella, Stenotrophomonas, Staphyloccocus, Streptococcus,Synechococcus, Vibrio, and Yersina; or anerobic bacterial genera such asPeptostreptococci, Porphyromonas, Actinomyces, Clostridium, Bacteroides,Prevotella, Anaerobiospirillum, Fusobacterium, and Bilophila. In someembodiments, the subject having an NK cell-responsive disorder can be inneed of treatment for a bacterial infection from Allochromatium vinosum,Acinetobacter baumanii, Bacillus anthracis, Campylobacter jejuni,Chlamydia trachomatis, Chlamydia pneumoniae, Clostridium spp.,Citrobacter spp., Escherichia coli, Enterobacter spp., Enterococcusfaecalis, Enterococcus faecium, Francisella tularensis, Haemophilusinfluenzae, Helicobacter pylori, Klebsiella spp, Listeria monocytogenes,Moraxella catarrhalis, Mycobacterium tuberculosis, Neisseriameningitidis, Neisseria gonorrhoeae, Proteus mirabilis, Proteusvulgaris, Pseudomonas aeruginosa, Salmonella spp., Serratia spp.,Shigella spp., Stenotrophomonas maltophilia, Staphyloccocus aureus,Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcuspyogenes, Streptococcus agalactiae, Yersina pestis, and Yersinaenterocolitica, or the like; or Peptostreptococci asaccharolyticus,Peptostreptococci magnus, Peptostreptococci micros, Peptostreptococciprevotii, Porphyromonas asaccharolytica, Porphyromonas canoris,Porphyromonas gingivalis, Porphyromonas macaccae, Actinomyces israelii,Actinomyces odontolyticus, Clostridium innocuum, Clostridiumclostridioforme, Clostridium difficile, Bacteroides tectum, Bacteroidesureolyticus, Bacteroides gracilis (Campylobacter gracilis), Prevotellaintermedia, Prevotella heparinolytica, Prevotella oris-buccae,Prevotella bivia, Prevotella melaninogenica, Fusobacterium naviforme,Fusobacterium necrophorum, Fusobacterium varium, Fusobacterium ulcerans,Fusobacterium russii, Bilophila wadsworthia, Haemophilus ducreyi;Calymmatobacterium granulomatis, or the like.

Compounds or pharmaceutical compositions or the invention can beparticularly useful for treating a subject with an intracellularinfection. It is generally believed in the art that NK cells areparticularly effective against intracellular infections. Intracellularinfections are those wherein a portion of the infecting pathogen resideswithin cells of the subject.

For example, intracellular infections can be caused by one or morebacteria selected from: Ehrlichia (e.g., obligate, intracellularbacteria that can appear as small cytoplasmic inclusions in lymphocytesand neutrophils such as Ehrlichia sennetsu, Ehrlichia canis, Ehrlichiachaffeensis, Ehrlichia phagocytophilia, or the like); Listeria (e.g.,Listeria monocytogenes); Legionella (e.g., Legionella pneumophila);Rickettsiae (e.g., Rickettsiae prowazekii, Rickettsiae typhi(Rickettsiae mooseri), Rickettsiae rickettsii, Rickettsiaetsutsugamushi, Rickettsiae sibirica; Rickettsiae australis; Rickettsiaeconorii; Rickettsiae akari; Rickettsiae burnetii); Chlamydia (e.g.,Chlamydia psittaci; Chlamydia pneumoniae; Chlamydia trachomatis, or thelike); Mycobacterium (Mycobacterium tuberculosis; Mycobacterium marinum;Mycobacterium Avium Complex; Mycobacterium bovis; Mycobacteriumscrofulaceum; Mycobacterium ulcerans; Mycobacterium leprae (Leprosy,Hansen's Bacillus)); Brucella (e.g., Brucella melitensis; Brucellaabortus; Brucella suis; Brucella canis); genus Coxiella (e.g., Coxiellaburnetii); or the like. Thus, in some embodiments, the subject can havean intracellular bacterial infection caused by a bacterium selected fromthe genera Ehrlichia: Listeria; Legionella; Rickettsiae; Chlamydia;Mycobacterium; Brucella; and Coxiella.

In various embodiments, the subject having an NK cell-responsivedisorder can be in need of treatment for a bacterial infection from oneor more upper respiratory tract bacteria. Examples of upper respiratorytract bacteria include those belonging genera such as Legionella,Pseudomonas, and the like. In some embodiments, the bacteria can bePseudomonas aeruginosa. In particular embodiments, the bacteria can beLegionella pneumophila (e.g., including serogroups 1, 2, 3, 4, 5, 6, 7,8, and the like), Legionella dumoffli, Legionella longbeacheae,Legionella micdadei, Legionella oakridgensis, Legionella feelei,Legionella anisa, Legionella sainthelensi, Legionella bozemanii,Legionella gormanii, Legionella wadsworthii, Legionella jordanis, orLegionella gormanii.

In some embodiments, the subject having an NK cell-responsive disordercan be in need of treatment for a bacterial infection from one thatcauses acute bacterial exacerbation of chronic bronchitis (ABECB) in thesubject. Typically, ABECB can be caused by Streptococcus pneumoniae,Haemophilus influenzae, Haemophilus parainfluenzae, or Moraxellacatarrhalis.

In some embodiments, the subject having an NK cell-responsive disordercan be in need of treatment for a bacterial infection from one thatcauses acute community acquired pneumonia (CAP) in the subject.Typically, CAP can be caused by Streptococcus pneumoniae, Haemophilusinfluenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, Chlamydiapneumoniae, or Klebsiella pneumoniae. In a particular embodiment, theCAP can be caused by drug resistant bacteria, e.g., a multi-drugresistant strain of Streptococcus pneumoniae.

In various embodiments, the subject having an NK cell-responsivedisorder can be in need of treatment for a bacterial infection fromStreptococcus pneumoniae, Haemophilus influenzae, Haemophilusparainfluenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, Chlamydiapneumoniae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcuspyogenes, Acinetobacter lwoffi, Klebsiella oxytoca, Legionellapneumophila, or Proteus vulgaris.

In various embodiments, the subject having an NK cell-responsivedisorder can be in need of treatment for a bacterial infection frommaxillary sinus pathogenic bacteria. As used herein, maxillary sinuspathogenic bacteria is a bacterial strain isolated from acute or chronicmaxillary sinusitis, or, for example, a maxillary sinus isolate ofStaphylococcus aureus, Streptococcus pneumoniae, Haemophilus spp.,Moraxella catarrhalis, an anaerobic strain of non-fermentative Gramnegative bacilli, Neisseria meningitides or β-haemolytic Streptococcus.In various embodiments, maxillary sinus pathogenic bacteria can includea bacterial strain isolated from acute or chronic maxillary sinusitis; amaxillary sinus isolate of Staphylococcus aureus, Streptococcuspneumoniae, Haemophilus spp., Moraxella catarrhalis, an anaerobic strainof non-fermentative Gram negative bacilli, Neisseria meningitidis,β-haemolytic Streptococcus, Haemophilus influenzae, anEnterobacteriaccae, a non-fermentative Gram negative bacilli.Streptococcus pneumoniae, Streptococcus pyogenes, amethicillin-resistant Staphylococcus spp., Legionella pneumophila,Mycoplasma spp. and Chlamydia spp., Haemophilus influenzae, Haemophilusparainfluenzae, Peptostreptococcus, Bacteroides spp., and Bacteroidesurealyticus.

In various embodiments, the subject having an NK cell-responsivedisorder can be in need of treatment for a bacterial infection thatcauses a urinary tract infection (UTI) in the subject. Examples of UTIsinclude urethritis, cystitis, prostatitis, pyclonephritis (acute,chronic, and xanthogranulomatous), and hematogenous UTI (e.g., frombacteremia with virulent bacilli such as Salmonella, Staphylococcusaureus, and the like). Typically, UTIs can be caused by gram-negativeaerobic bacteria, e.g., Escherichia (e.g., Escherichia coli),Klebsiella, Proteus, Enterobacter, Pseudomonas, and Serratia;gram-negative anaerobic bacteria; gram-positive bacteria, e.g.,Enterococci (e.g., Enterococcus faecalis) and Staphylococcus sp (e.g.,Staphylococcus saprophyticus, Staphylococcus aureus, and the like);Mycobacterium tuberculosis; and sexually transmitted bacterialinfections (e.g., Chlamydia trachomatis, Neisseria gonorrhoeae, and thelike).

In certain embodiments, the subject having an NK cell-responsivedisorder can be in need of treatment for infections from microorganismsthat cause sexually transmitted diseases, for example, Treponemapallidum; Trichomonas vaginalis; Candicha (Candida albicans); Neisseriagonorrhoeae; Chlamydia trachomatis; Mycoplasma genitalium, Ureaplasmaurealyticum; Haemophilus ducreyi; Calymmatobacterium granolomatis(formerly Donovania granulomatis); herpes simplex viruses (HSV-1 orHSV-2), human papillomavirus [HPV]; human immunodeficiency virus (HIV);various bacterial (Shigella, Campylobacter, or Salmonella), viral(hepatitis A), or parasitic (Giardia or amoeba, e.g., Entamoeba dispar(previously Entamoeba histolytica); or the like.

Thus, in various embodiments, the subject having an NK cell-responsivedisorder can be in need of treatment for an infection resulting in upperrespiratory tract bacterial infection, acute bacterial exacerbation ofchronic bronchitis; acute community acquired pneumonia, maxillary sinuspathogenic bacteria; a urinary tract infection; or a sexuallytransmitted infection.

The compounds and pharmaceutical compositions of the present inventioncan be particularly effective for treating a subject with a viralinfection. Thus, in various embodiments, a subject having an NKcell-responsive disorder can be in need of treatment for infection fromviruses such as Picornaviruses (e.g., Polio Virus, rhinoviruses andcertain echoviruses and coxsackieviruses); Parvoviridae (HumanParvovirus B19); Hepatitis, e.g., Hepadnavirus (Hepatitis B);Papovavirus (JC Virus); Adenovirus (Human Adenovirus); Herpesvirus(e.g., Cytomegalovirus, Epstein Barr Virus (Mononucleosis),Mononucleosis-Like Syndrome, Roseola Infantum, Varicella Zoster Virus(Chicken Pox), Herpes Zosier (Shingles), Herpes Simplex Virus (OralHerpes, Genital Herpes)), Poxvirus Smallpox); Calicivirus (NorwalkVirus), Arbovirus (e.g., Togavirus (Rubella virus, Dengue virus),Flavivirus (Yellow Fever virus), Bunyavirus (California EncephalitisVirus), Reovirus (Rotavirus)); Coronavirus (Coronavirus); Retrovirus(Human Immunodeficiency Virus 1, Human Immunodeficiency Virus 2);Rhabdovirus (Rabies Virus), Filovirus (Marburg Virus, Ebola virus, otherhemorrhagic viral diseases); Paramyxovirus (Measles Virus, Mumps Virus);Orthomyxovirus (Influenza Virus), Arenavirus (Lassa Fever); human T-cellLymphotrophic virus type I and II (HTLV-I, HTLV II); humanpapillomavirus [HPV]; or the like. Thus, in various embodiments, thesubject can have an infection caused by a virus selected fromPicornavirus; Parvoviridae; Hepatitis virus; Papovavirus; Adenovirus;Herpesvirus, Poxvirus; Calicivirus, Arbovirus; Coronavirus; aRetrovirus; Rhabdovirus; Paramyxovirus; Orthomyxovirus; Arenavirus;human T-cell Lymphotrophic virus; human papillomavirus; and humanimmunodeficiency virus.

In some embodiments, a subject having an NK cell-responsive disorder canbe in need of treatment for an infection from a virus or an infectionthereof such as human immunodeficiency virus-1, human immunodeficiencyvirus-2, Cytomegalovirus, Epstein Barr Virus, Mononucleosis-LikeSyndrome, Roscola Infantum, Varicella Zoster Virus, Herpes Zoster,Herpes Simplex Virus, or hepatitis.

It is believed that the methods can be particularly effective fortreating a subject with a parasitic infection. Thus, in variousembodiments, a subject having an NK cell-responsive disorder can be inneed of treatment for an infection from Plasmodia (e.g., Plasmodiafalciparum, Plasmodia vivax, Plasmodia ovale, and Plasmodia malariae,typically transmitted by anopheline mosquitoes); Leishmania (transmittedby sandflies and caused by obligate intracellular protozoa, e.g.,Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmaniamexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmaniatropica; Leishmania major; Leishmania aethiopica; and the subgenusViannia, Leishmania Viannia braziliensis, Leishmania Viannia guyanensis,Leishmania Viannia panamensis, and Leishmania Viannia peruviana),Trypanosoma (e.g., sleeping sickness caused by Trypanosoma bruceigambiense, and Trypanosoma brucei rhodesiense); amoebas of the generaNacgleria or Acanthamoeba; pathogens such as genus Entamoeba (Entamoebahistolytica and Entamoeba dispar); Giardia lamblia; Cryptosporidium;Isospora; Cyclospora, Microsporidia, Ascaris lumbricoides; infectionwith blood flukes of the genus Schistosoma (e.g.; S. haematobium; S.mansoni; S. japonicum; S. mekongi; S. intercalatum); Toxoplasmosis(e.g., Toxoplasma gondii); Treponema pallidum; Trichomonas vaginalis; orthe like.

In some embodiments, the subject having an NK cell-responsive disordercan have an infection caused by a protozoa selected from Toxoplasmagondii, Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense,Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmaniamexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmaniatropica; Leishmania major; Leishmania aethiopica; and the subgenusViannia, Leishmania Viannia braziliensis, Leishmania Viannia guyanensis,Leishmania Viannia panamensis, Leishmania Viannia peruviana, Plasmodiafalciparum, Plasmodia vivax, Plasmodia ovale, and Plasmodia malariae.

In the last century, antibiotics were developed that led to significantreductions in mortality. Unfortunately, widespread use has led to therise of antibiotic resistant bacteria, e.g., methicillin resistantStaphyloccocus aureus (MRSA), vancomycin resistant enterococci (VRE),and penicillin-resistant Streptococcus pneumoniae (PRSP). Some bacteriaare resistant to a range of antibiotics, e.g., strains of Mycobacteriumtuberculosis resist isoniazid, rifampin, ethambutol, streptomycin,ethionamide, kanamycin, and rifabutin. In addition to resistance, globaltravel has spread relatively unknown bacteria from isolated areas to newpopulations. Furthermore, there is the threat of bacteria as biologicalweapons. These bacteria may not be easily treated with existingantibiotics.

The compounds or the pharmaceutical compositions of the invention can beparticularly effective for treating a subject for drug-resistantpathogens, for example, drug resistant bacteria, or pathogens for whichno drugs are available, e.g., many viruses. Without wishing to be boundby theory, it is believed that because the compounds of the inventioncan act by increasing NK cell activity, and thus the NK cells can killinfective microorganisms or infected cells separately from any directaction of the compounds on the pathogen or infected cells. Thus, it isbelieved that the compounds of the invention can have at least one modeof action that is separate from typical anti-infective drugs such asantibiotics which can typically act directly on the bacteria themselves.

Drug resistant pathogens can be resistant to at least one and typicallymultiple agents, for example, drug resistant bacteria can be resistantto one antibiotic, or typically at least two antibiotics such aspenicillin, Methicillin, second generation cephalosporins (e.g.,cefuroxime, and the like), macrolides, tetracyclines,trimethoprim/methoxazole, vancomycin, or the like. For example, in someembodiments, a subject can be treated for bacteria selected from astrain of multiple drug resistant Streptococcus pneumniae (MDRSP,previously known as penicillin resistant Streptococcus pneumoniae,PRSP), vancomycin resistant Enterococcus, methicillin resistantStaphylococcus Aureus, penicillin resistant Pneumococcus, antibioticresistant Salmonella, resistant and multi-resistant Neisseria Gonorrhea(e.g., resistant to one, two or more of tetracycline, penicillin,fluoroquinolones, cephalosporins, ceftriaxone (Rocephin), Cefixime(Suprax), Azithromycin, or the like), and resistant and multi-resistantTuberculosis (e.g., resistant to one, two or more of Isoniazid,Rifampin, Ethambutol, Pyrazinamide, Aminoglycoside, Capreomycin,Ciprofloxacin, Ofloxacin, gemifloxacin, Cycloserine, Ethionamide,para-aminosalicylic acid or the like).

In some embodiments, NK cell activity can be increased in subjects thathave an immunodeficiency. In various embodiments, this can be due todecreased or deficient NK cell activity. In some embodiments, theimmunodeficiency can be any known immunodeficiency, even those that donot directly impact NK cells. Without wishing to be bound by theory, itis believed that boosting NK cell activity can augment immune functionin many immunodeficiency conditions to “make-up” at least in part, foraspects of immunodeficiency separate from those aspects directlyconcerned with NK cell activity.

ln various embodiments, immunodeficiency disorders can include disorderswith increased susceptibility to infection, for example, one or moredisorders selected from: circulatory and systemic disorders (sickle celldisease, diabetes mellitus, nephrosis, varicose veins, congenitalcardiac defects); obstructive disorders (ureteral or urethral stenosis,bronchial asthma, bronchiectasis, allergic rhinitis, blocked Eustachiantubes); integumentary defects (eczema, burns, skull fractures, midlinesinus tracts, ciliary abnormalities); primary immunodeficiencies(X-linked agammaglobulinemia, DiGeorge anomaly, chronic granulomatousdisease, C3 deficiency); secondary immunodeficiencies (malnutrition,prematurity, lymphoma, splenectomy, uremia, immunosuppressive therapy,protein-losing enteropathy, chronic viral discases); unusualmicrobiologic factors (antibiotic overgrowth, chronic infections withresistant organism, continuous reinfection (contaminated water supply,infectious contact, contaminated inhalation therapy equipment)); foreignbodies, trauma (ventricular shunts, central venous catheter, artificialheart valves, urinary catheter, aspirated foreign bodies) allogeneictransplant, graft-versus-host disease, uterine dysfunction (e.g.,endometriosis), or the like.

In various embodiments, immunodeficiency disorders can include forexample, transient hypogammaglobulinemia of infancy, selective IgAdeficiency, X-linked agammaglobulinemian (Bruton's Agammaglobulinemia,Congenital Agammaglobulinemia), common variable immunodeficiency(Acquired Agammaglobulinemia), hyper-IgM immunodeficiency, IgG subclassdeficiency, chronic mucocutaneous Candidiasis, combinedimmunodeficiency, Wiskott-Aldrich syndrome, ataxia-telangiectasia,X-linked lymphoproliferative syndrome, hyper-IgE syndrome (Job-BuckleySyndrome), chronic granulotomatous disease, leukocyte adhesiondeficiency (MAC-I/LFA-1/CR3 deficiency), or the like.

In various embodiments, immunodeficiency disorders can include primaryimmunodeficiency disorders for example: B-cell (antibody) deficiencies(X-linked agammaglobulinemia; Ig deficiency with hyper-IgM (XL); IgAdeficiency); IgG subclass deficiencies, Antibody deficiency with normalor elevated Igs, Immunodeficiency with theymoma, Common variableimmunodeficiency, Transient hypogammaglobulinemia of infancy); T-cell(cellular) deficiencies (Predominant T-cell deficiency: DiGeorgeanomaly, Chronic mucocutancous candidiasis. Combined immunodeficiencywith Igs (Nezelof syndrome), Nucleoside phosphorylase deficiency (AR),Natural killer cell deficiency, Idiopathic CD4 lymphocytopenia, CombinedT- and B-cell deficiencies: Severe combined immunodeficiency (AR or XL).Adenosine deaminase deficiency (AR), Reticular dysgenesis, Barelymphocyte syndrome, Ataxia-telangiectasia (AR), Wiskott-Aldrichsyndrome (XL), Short-limbed dwarfism, XL lymphoproliferative syndrome);Phagocytic disorders (Defects of cell movement: HyperimmunoglobulinemiaE syndrome, Leukocyte adhesion defect type 1 (AR), Defects ofmicrobicidal activity: Chronic granulomatous disease (XL or AR),Neutrophil G6PD deficiency, Myeloperoxidase deficiency (AR).Chediak-Higashi syndrome (AR)); Complement disorders (Defects ofcomplement components: C1q deficiency, Defects of control proteins: C1inhibitor deficiency (D1), Factor (C3b inactivator) deficiency (ACD),Factor H deficiency (ACD), Factor D deficiency (ACD), Properdindeficiency (XL)); or the like.

In various embodiments, immunodeficiency disorders can include secondaryimmunodeficiency disorders, for example, one or more conditions selectedfrom: Premature and newborn infants (Physiologic immunodeficiency due toimmaturity of immune system); Hereditary and metabolic diseases(Chromosome abnormalities (e.g., Down syndrome), Uremia, Diabetes (i.e.,complications from diabetes such as gangrene associated with peripheralcirculatory and nerve dysfunction), Malnutrition, Vitamin and mineraldeficiencies, Protein-losing enteropathies; Nephrotic syndrome, Myotonicdystrophy, Sickle cell disease); Immunosuppressive agents (Radiation,Immunosuppressive drugs, Corticosteroids, Anti-lymphocyte oranti-thymocyte globulin, Anti-T-cell monoclonal antibodies); Infectiousdiseases (Congenital rubella, Viral exanthems (e.g., measles,varicella), HIV infection, Cytomegalovirus infection, Infectiousmononucleosis, Acute bacterial disease, Severe mycobacterial or fungaldisease); Infiltrative and hematologic diseases (Histiocytosis,Sarcoidosis, Hodgkin's disease and lymphoma, Leukemia, Myeloma,Agranulocytosis and aplastic anemia); Surgery and trauma (Burns,Splenectomy, Anesthesia, wounds); and Miscellaneous (SLE, Chronic activehepatitis, Alcoholic cirrhosis, Aging, Anticonvulsive drugs,Graft-vs.-host disease); or the like.

In certain embodiments, the subject having an NK cell-responsivedisorder can be in need of treatment for burns or wounds. Typically,such a wound or burn is a severe injury that places a significant burdenon the subject's immune defenses. For example, in some embodiments, thesubject is treated for a second or third degree burn covering at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, or more of the surfacearea of the subject's body. Also, in some embodiments, the subject istreated for a wound or wounds, e.g., an open wound of at least about 1cm², 2 cm², 5 cm², 10 cm², 20 cm², 50 cm² or larger, or 1%, 2%, 3%, 4%,5%, 10%, 15%, or more of the surface area of the subject's body; or oneor more incisions penetrating the skin totaling at least 1 cm, 2 cm, 3cm, 4 cm, 5 cm, 7 cm, 10 cm, 20 cm, 25 cm, 50 cm in length; anamputation; and the like.

In various embodiments, the subject having an NK cell-responsivedisorder can have an infection caused by antibiotic resistant bacteria.In some embodiments, the subject can have an infection caused by abacterium selected from multiple drug resistant Streptococcuspneumoniae, vancomycin resistant Enterococcus, methicillin resistantStaphylococcus Aureus, penicillin resistant Pneumococcus, antibioticresistant Salmonella, resistant/multi-resistant Neisseria Gonorrhea, andresistant/multi-resistant Tuberculosis. In some embodiments, the subjectcan have a bacterial infection resistant to at least one antibioticselected from penicillin, Methicillin, second generation cephalosporins,macrolides, tetracyclines, trimethoprim/methoxazole, vancomycin,tetracycline, fluoroquinolones, ceftriaxone, Cefixime, Azithromycin,Isoniazid, Rifampin, Ethambutol, Pyrazinamide, Aminoglycoside,Capreomycin, Ciprofloxacin, Ofloxacin, gemifloxacin, Cycloserine,Ethionamide, and para-aminosalicylic acid.

Thus, various embodiments, the subject having an NK cell responsivedisorder can have an immunodeficiency disorder. In some embodiments, thesubject can have a primary immunodeficiency disorder. In someembodiments, the subject can have a secondary immunodeficiency disorder.

In some embodiments, immunodeficiency disorders can include uremia,diabetes (infective complications thereof, malnutrition, vitamin andmineral deficiencies, protein-losing enteropathies, nephrotic syndrome,myotonic dystrophy, sickle cell disease; or the like.

In some embodiments, immunodeficiency disorders can be cause or bepartially caused by immunosuppressive agents, e.g., radiation,immunosuppressive drugs, corticosteroids, anti-lymphocyte oranti-thymocyte globulin, anti-T-cell monoclonal antibodies; or the like.

In some embodiments, immunodeficiency disorders can caused or partiallycaused by surgery and trauma, e.g., burns, splenectomy, anesthesia,wounds, implanted medical devices; or the like.

In some embodiments, immunodeficiency disorders can include chronicfatigue syndrome (chronic fatigue immune dysfunction syndrome);Epstein-Barr virus infection, post viral fatigue syndrome,post-transplantation syndrome (host-graft disease), exposure to nitricoxide synthase inhibitors, aging, severe combined immunodeficiency,variable immunodeficiency syndrome, and the like.

Increasing NK cell activity would also be beneficial for treatingsubjects with disorders including, but not limited to aneurodegenerative disorder. As used herein, a neurodegenerative disorderinvolves degradation of neurons such as cerebral, spinal, and peripheralneurons (e.g., at neuromuscular junctions), more typically degradationof cerebral and spinal neurons. Neurodegenerative disorders can includeAlzheimer's disease; Huntington's disease; Parkinson's disease;spinal/bulbar muscular atrophy (e.g., Kennedy's disease),spinocerebellar ataxic disorders, and other neuromuscular atrophies;familial amyotrophic lateral sclerosis; ischemia; seizure; hypothermia;hyperthermia; burn trauma; atherosclerosis; radiation exposure;glaucoma; toxin exposure; mechanical injury; inflammation; epilepticseizure, injury-induced seizure, chemically-induced seizure, or otherdiseases associated with superoxide dismutase (SOD) mutations; and thelike. Neurodegenerative disorders can also include degradation ofneurons caused by ischemia, seizure, thermal stress, radiation, toxinexposure, infection, injury, and the like. Ischemia can damage tissuethrough multiple routes, including oxygen depletion, glucose depletion,oxidative stress upon reperfusion, and/or glutamate toxicity, and thelike. Ischemia can result from an endogenous condition (e.g., stroke,heart attack, and the like), from accidental mechanical injury, fromsurgical injury (e.g., reperfusion stress on transplanted organs), andthe like. Alternatively, tissues that can be damaged by ischemia includeneurons, cardiac muscle, liver tissue, skeletal muscle, kidney tissue,pulmonary tissue, pancreatic tissue, and the like.

Other disorders in which increasing NK cell activity would be beneficialinclude disorders due to thermal stress, (thermal stress includeshyperthermia (e.g., from fever, heat stroke, burns, and the like) andhypothermia); radiation damage, e.g., due to visible light, ultravioletlight, microwaves, cosmic rays, alpha radiation, beta radiation, gammaradiation, X-rays, and the like, (for example, the damage could beradiation damage to non-cancerous tissue in a subject treated for cancerby radiation therapy); mechanical injury, e.g., trauma from surgery,accidents, certain disease conditions (e.g., pressure damage inglaucoma) and the like; and exposure to a toxin, e.g., exposure to aneurotoxin selected from methamphetamine; antiretroviral HIVtherapeutics (e.g., nucleoside reverse transcriptase inhibitors; heavymetals (e.g., mercury, lead, arsenic, cadmium, compounds thereof, andthe like), amino acid analogs, chemical oxidants, ethanol, glutamate,metabolic inhibitors, antibiotics, and the like.

In some embodiments, the invention provides a method for treating orinhibiting angiogenesis in a subject in need thereof, comprisingadministering to the subject an effective amount of a compound asdescribed herein. As used herein, the term “angiogenesis” refers to afundamental process of generating new blood vessels in tissues ororgans. Angiogenesis is involved with or associated with many diseasesor conditions, including, but not limited to: cancer; ocular neovasculardisease; age-related macular degeneration; diabetic retinopathy,retinopathy of prematurity; conical graft rejection; neovascularglaucoma; retrolental fibroplasias; epidemic keratoconjunctivitis;Vitamin A deficiency; contact lens overwear; atopic keratitis; superiorlimbic keratitis; pterygium keratitis sicca; sjogrens; acne rosacea;warts; eczema; phylectenulosis: syphilis; Mycobacteria infections; lipiddegeneration; chemical burns; bacterial ulcers; fungal ulcers; Herpessimplex infections; Herpes zoster infections; protozoan infections;Kaposi's sarcoma; Mooren's ulcer; Terrien's marginal degeneration;mariginal keratolysis; rheumatoid arthritis; systemic lupus;polyarteritis; trauma; Wegener's sarcoidosis: scleritis; Stevens-johnsondisease; pemphigoid; radial keratotomy; corneal graph rejection;diabetic retinopathy; macular degeneration; sickle cell anemia; sarcoid;syphilis; pseudoxanthoma elasticum; Paget's disease; vein occlusion;artery occlusion; carotid obstructive disease; chronic uveitis/vitritis;mycobacterial infections; Lyme's disease; systemic lupus erythematosis;retinopathy of prematurity; Eales' disease; Behcet's disease; infectionscausing a retinitis or choroiditis; presumed ocular histoplasmosis;Best's disease; myopia; optic pits; Stargardt's disease; pars planitis;chronic retinal detachment; hyperviscosity syndromes; toxoplasmosis;trauma and post-laser complications; diseases associated with rubeosis(neovasculariation of the angle); diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue including all forms ofproliferative vitreorctinopathy; rheumatoid arthritis; osteoarthritis;ulcerative colitis; Crohn's disease; Bartonellosis; atherosclerosis;Osler-Weber-Rendu disease; hereditary hemorrhagic telangiectasia;pulmonary hemangiomatosis; pre-eclampsia; endometriosis; fibrosis of theliver and of the kidney; developmental abnormalities (organogenesis);skin disclolorations (e.g., hemangioma, nevus flammeus, or nevussimplex); wound healing; hypertrophic scars, i.e., keloids; woundgranulation; vascular adhesions; cat scratch disease (Roche ninaliaquintosa); ulcers (Helicobacter pylori); keratoconjunctivitis;gingivitis; periodontal disease; epulis; hepatitis; tonsillitis;obesity; rhinitis; laryngitis; tracheitis; bronchitis; bronchiolitis;pneumonia; interstitial pulmonary fibrosis, pulmonary edema;neurodermitis; thyroiditis; thyroid enlargement; endometriosis;glomerulonephrtis; gastritis; inflammatory bone and cartilagedestruction; thromboembolic disease; and Buerger's disease.Anti-angiogenesis can be demonstrated by any method known to thoseskilled in the art, such as the method described herein in Examples 10and 11.

A “subject” is a mammal, preferably a human, but can also be an animalin need of veterinary treatment, e.g., companion animals (e.g., dogs,cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, andthe like) and laboratory animals (e.g., rats, mice, guinea pigs, and thelike).

Suitable pharmaceutically acceptable carriers or diluents may containinert ingredients which do not inhibit the biological activity of thecompounds described herein. The pharmaceutically acceptable carriers ordiluents should be biocompatible, i.e., non-toxic, non-inflammatory,non-immunogenic and devoid of other undesired reactions upon theadministration to a subject. Standard pharmaceutical formulationtechniques can be employed, such as those described in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa.Formulation of the compound to be administered will vary according tothe route of administration selected (e.g., solution, emulsion,capsule). Suitable pharmaceutical carriers for parenteral administrationinclude, for example, sterile water, physiological saline,bacteriostatic saline (saline containing about 0.9% mg/ml benzylalcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactateand the like. Methods for encapsulating compositions such as in acoating of hard gelatin or cyclodextrins) are known in the art (Baker,et al., “Controlled Release of Biological Active Agents”, John Wiley andSons, 1986).

An “effective amount” is the quantity of compound in which a beneficialclinical outcome is achieved when the compound is administered to asubject. For example, when a compound of the invention is administeredto a subject with a cancer, a “beneficial clinical outcome” includes areduction in tumor mass, a reduction in metastasis, a reduction in theseverity of the symptoms associated with the cancer and/or an increasein the longevity of the subject compared with the absence of thetreatment. When a compound of the invention is administered to a subjectwith a an Hsp70-responsive disorder or an NK cell-responsive disorder, a“beneficial clinical outcome” includes reduction in the severity ornumber of symptoms associated with the disorder, elimination of aninfection, or increase in the longevity of the subject compared with theabsence of the treatment. The precise amount of compound administered toa subject will depend on the type and severity of the disease orcondition and on the characteristics of the subject, such as generalhealth, age, sex, body weight and tolerance to drugs. It may also dependon the degree, severity and type of cancer. The skilled artisan will beable to determine appropriate dosages depending on these and otherfactors. Effective amounts of the disclosed compounds typically rangebetween about 1 mg/mm² per day and about 10 grams/mm² per day, andpreferably between 10 mg/mm² per day and about 5 grams/mm². In someembodiments, effective amounts of the disclosed compounds includemicrogram to milligram amounts of the compound per kilogram of subjector sample weight (e.g., about 1 μg/kg to about 500 mg/kg, about 500μg/kg to about 250 mg/kg, about 1 mg/kg to about 100 mg/kg, about 10mg/kg to about 50 mg/kg, and the like). When co-administered withanother anti-cancer agent for the treatment of cancer, an “effectiveamount” of the second anti-cancer agent will depend on the type of drugused. Suitable dosages are known for approved anti-cancer agents and canbe adjusted by the skilled artisan according to the condition of thesubject, the type of cancer being treated and the compound of theinvention being used.

The compounds and pharmaceutical compositions disclosed herein isadministered by any suitable route, including, for example, orally incapsules, suspensions or tablets or by parenteral administration.Parenteral administration can include, for example, systemicadministration, such as by intramuscular, intravenous, subcutaneous, orintraperitoneal injection. The compound and pharmaceutical compositiondisclosed herein can also be administered orally (e.g., dietary),topically, by inhalation (e.g., intrabronchial, intranasal, oralinhalation or intranasal drops), or rectally, depending on the type ofcancer to be treated. Oral and parenteral administrations are preferredmodes of administration.

The present invention is illustrated by the following examples, whichare not intended to be limiting in any way.

EXEMPLIFICATION Example 1 Synthesis of the Compounds

Synthesis of Compound I

Copper (II) chloride (277 mg, 2.0 mmol) was added to a solution ofbis[thiohydrazide amide] A (800 mg, 2.0 mmol) in EtOH (10.0 mL). Themixture was stirred at room temperature for 20 minutes. Water was thenadded. The solid was collected by filtration. The solid was taken up inmethylene chloride. The resulting solution was washed with water (2×),dried (Na₂SO₄), filtered and concentrated to give crude solid. The solidwas washed with acetone to give the pure compound 1 (600 mg). Singlecrystal solid were obtained by recrystallization from acetonitrile. MS(ESI) [M+H⁺]: 462. MP; 198-202° C. (decomposed). Anal. calc. ForC₁₉H₁₈CuN₄O₂S₂: C, 49.39; H, 3.93; N, 12.13. found: C, 49.36; H, 3.68;N, 11.92.

Table of Bond Distances in Armstrongs for Compound 1 Atom 1 Atom 2Distance Cu1 N7 1.944(2) Cu1 N3 1.948(2) Cu1 S1 2.2346(7) Cu1 S92.2375(7) S1 C1 1.694(3) S9 C9 1.707(3) O4 C4 1.231(3) O6 C6 1.227(3) N2C1 1.321(3) N2 N3 1.405(3) N2 C21 1.482(3) N3 C4 1.370(3) N7 C6 1.363(3)N7 N8 1.414(3) N8 C9 1.316(3) N8 C81 1.481(3) C1 C11 1.479(4) C4 C51.510(4) C5 C6 1.519(4) C5 H51 0.94(4) C5 H52 1.01(3) C9 C91 1.481(4)C11 C12 1.392(3) C11 C16 1.394(3) C12 C13 1.383(4) C12 H12 0.950 C13 C141.389(4) C13 H13 0.950 C14 C15 1.389(4) C14 H14 0.950 C15 C16 1.384(4)C15 H15 0.950 C16 H16 0.950 C21 H21A 0.980 C21 H21B 0.980 C21 H21C 0.980C81 H81A 0.980 C81 H81B 0.980 C81 H81C 0.980 C91 C92 1.391(4) C91 C961.403(3) C92 C93 1.388(4) C92 H92 0.950 C93 C94 1.382(4) C93 H93 0.950C94 C95 1.393(4) C94 H94 0.950 C95 C96 1.384(4) C95 H95 0.950 C96 H960.950 Numbers in parentheses are estimated standard deviations in theleast significant digits.

Table of Bond Angles in Degrees for Compound 1 Atom 1 Atom 2 Atom 3Angle N7 Cul N3 96.27(8) N7 Cul S1 161.69(6) N3 Cul S1 86.92(6) N7 CulS9 86.58(6) N3 Cul S9 158.38(6) S1 Cul S9 97.10(3) C1 S1 Cul 96.53(9) C9S9 Cul 96.97(9) C1 N2 N3 118.7(2) C1 N2 C21 122.7(2) N3 N2 C21116.67(19) C4 N3 N2 112.6(2) C4 N3 Cul 118.96(18) N2 N3 Cul 115.96(14)C6 N7 N8 113.3(2) C6 N7 Cul 120.48(17) C8 N7 Cul 116.52(14) C9 N8 N7118.6(2) C9 N8 C81 123.2(2) N7 N8 C81 115.93(19) N2 C1 C11 120.7(2) N2C1 S1 121.3(2) C11 C1 S1 118.07(17) O4 C4 N3 125.2(2) O4 C4 C5 119.7(2)N3 C4 C5 114.9(2) C4 C5 C6 124.5(2) C4 C5 H51 111(2) C6 C5 H51 106(2) C4C5 H52 101.6(19) C6 C5 H52 111.1(19) H51 C5 H52 99(3) O6 C6 N7 125.4(2)O6 C6 C5 118.8(2) N7 C6 C5 115.6(2) N8 C9 C91 121.6(2) N8 C9 S9 120.7(2)C91 C9 S9 117.68(18) C12 C11 C16 119.6(2) C12 C11 C1 119.8(2) C16 C11 C1120.5(2) C13 C12 C11 119.7(2) C13 C12 H12 120.20 C11 C12 H12 120.20 C12C13 C14 120.7(3) C12 C13 H13 119.60 C14 C13 H13 119.60 C13 C14 C15119.6(3) C13 C14 H14 120.20 C15 C14 H14 120.20 C16 C15 C14 119.9(2) C16C15 H15 120.00 C14 C15 H15 120.00 C15 C16 C11 120.4(2) C15 C16 H16119.80 C11 C16 H16 119.80 N2 C21 H21A 109.50 N2 C21 H21B 109.50 H21A C21H21B 109.50 N2 C21 H21C 109.50 H21A C21 H21C 109.50 H21B C21 H21C 109.50N8 C81 H81A 109.50 N8 C81 H81B 109.50 H81A C81 H81B 109.50 N8 C81 H81C109.50 H81A C81 H81C 109.50 H81B C81 H81C 109.50 C92 C91 C96 119.1(2)C92 C91 C9 121.6(2) C96 C91 C9 119.1(2) C93 C92 C91 120.6(2) C93 C92 H92119.70 C91 C92 H92 119.70 C94 C93 C92 120.1(3) C94 C93 H93 119.90 C92C93 H93 119.90 C93 C94 C95 119.8(3) C93 C94 H94 120.10 C95 C94 H94120.10 C96 C95 C94 120.5(2) C96 C95 H95 119.80 C94 C95 H95 119.80 C95C96 C91 119.9(3) C95 C96 H96 120.00 C91 C96 H96 120.00 Numbers inparentheses are estimated standard deviations in the least significantdigits.Synthesis of Compound 2

Compound 2 was prepared similarly as described for the preparation ofcompounds 1 using bis[thiohydrazide amide] A and nickel (II) chloridehexahydrate. MS (ESI) [M+H⁺]: 457. ¹H NMR (300 MHz, CDCl₃) δ 7.58-7.44(m, 10H), 3.61 (s, 6H), 3.59 (s, 2H).

Example 2 Biological Activity of Compound 1

M14 melanoma cells were seeded at 50,000 cells per well of 96-well platein 100 μl of Dulbecco's minimum essential medium (DMEM) supplementedwith 10% fetal bovine serum. Cells were cultured at 37° C. under 5%CO₂-95% air. After 16 hours of incubation, the test compound was addedto the cell culture. The compound was first diluted in 100% dimethylsulfoxide (DMSO) at 400-fold of the final concentrations actually usedin the assay. The DMSO solution was next diluted 20 fold with theculture medium, and then finally added to the assay wells at another20-fold dilution. Assay medium included the test compound withconcentrations as indicated with 0.25% DMSO. Cell viability was measuredwith a CCK8 assay (as described in the Technical Manual for CellCounting Kit-8, Product #CK04-11, CK04-13 and CK04-20, Dojindo MolecularTechnologies, Inc. MD, Tantular, I. S., et al. Tropical Medicine andInternational Health, 8(6), 569-574, 2003) in the last 15 minutes aftera 48 h incubation with the test compound. The data for compounds 1 and 2are compared with that of compound A, shown in FIG. 25. Each bar of thefigure expresses average percent inhibition against vehicle (0.25% DMSO)control (n=4). Error bars indicate standard deviation, The IC₅₀ valuesfor compound A compound 2 are greater than 12.5 μM; while the IC₅₀ valuefor compound 1 is 0.8 μM.

Examples 3-7

Heat shock proteins (Hsp) are induced under a variety of stressconditions and bind to other proteins to prevent their denaturation.Hsps can protect the cell from apoptotic death. Agents that induce theproduction of Hsp70 can have protective activity against a wide range ofinsults, and may have particular utility in neurological disorders. Theneuroprotectant activity of Hsp70 inducing compounds of the inventioncan be assessed in a variety of animal neurological disease models.Specifically, animal models of stroke, amyotrophic lateral sclerosis,Huntington's disease, Parkinson's disease, and Alzheimer's disease areappropriate settings for testing efficacy. Some example animal modelsare provided below.

Example 3 Cerebral Ischemia (Stroke)

The benefit of the disclosed treatment with Hsp70 inducing compounds ofthe invention can be assessed in rodent models of stroke. For examplethe stroke model described in Longa, et al. (Longa, E. Z., Weinstein, P.R., Carlson, S., and Cummins, R. (1989) Reversible middle cerebralartery occlusion without craniectomy in rats. Stroke 20:84-91) can beutilized.

Rats are anesthetized with ketamine, and then infarction is induced byextracranial vascular occlusion. A 4-0 nylon intraluminal suture isplaced into the cervical internal carotid artery and is advancedintracranially to block blood flow into the middle cerebral artery.Collateral blood flow is reduced by interrupting all branches of theexternal carotid artery and all extracranial branches of the internalcarotid artery. A compounds of the invention can be dosed just prior toor just after induction of the infarction. The dose may be, for example,10 to 100 mg/kg body weight administered once per week, three times perweek, or daily by any conventional mode of administration, e.g., orallyor intravenously. Neurologic deficit, mortality, gross pathology(infarction size), and histochemical staining can be analyzed to assessefficacy of the compounds. Since this is a very acute model, and deathis often observed by three days after infarction, the modeling mayconsist of only a single administration of drug.

Example 4 Familial Amyotrophic Lateral Sclerosis (ALS)

The efficacy of compounds of the invention in the treatment of ALS canbe modeled using the SOD1 transgenic mouse model (Gurney, M. E., Pu,Chiu, A. Y., Dal Canto, M. C., Polchow, C. Y., Alexander, D. D.,Caliendo, J., Hentati, A., Kwon, Y, W., and Deng, H. X. (1994) Motorneuron degeneration in mice that express a human CuZn superoxidedismutase mutation. Science 204:1772-1775). Mutations of human CuZnsuperoxide dismutase (SOD) are found in patients with familial ALS.Expression of the human SOD gene containing a substitution ofglycine-to-alanine at amino acid 93 leads to motor neuron disease intransgenic mice. As a result of motor neuron loss from the spinal cord,the mice became paralyzed and die by 5 to 6 months of age.

To test the efficacy of the Hsp70 inducing compounds of the invention,transgenic mice having the SOD1 mutation (SODI^(G93A)) are treated withthe compounds, and the effect on disease is monitored. The symptoms areclinically apparent in these animals at 2.5 to 3 months of age.Compounds can be dosed starting at this time. The dose may be, forexample, 10 to 100 mg/kg body weight administered once per week or threetimes per week by the oral or intravenous route. Endpoints includefunctional impairment of motor function as well as histological changes.The latter endpoints include histopathology of brain and spinal cordassessing degeneration of motor neurons and the appearance ofneurofilament-rich inclusions in spinal motor neurons. If long-termadministration is performed, the impact on mouse survival can beassessed.

Example 5 Huntington's Disease (HD)

A transgenic mouse model of HD exists, allowing the testing of Hsp70inducing compounds of the invention for efficacy in this disease setting(Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A.,Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S. W.,and Bates, G. P. (1996) Exon 1 of the HD gene with an expanded CAGrepeat is sufficient to cause a progressive neurological phenotype intransgenic mice. Cell 87:493-506; Carter, R. J., Lione, L. A. Humby, T.,Mangiarini, L., Mahal, A., Bates, G. P., Dunnett, S. B., and Morton, A.J. (1999) Characterization of progressive motor deficits in micetransgenic for the human Huntington's disease mutation. J. Neuroscience19:3248-3257). HD is caused by a CAG/polyglutamine repeat expansion.These transgenic mice (R6/2 transgenics) have the 5′ end of the human HDgene with (CAG)115-(CAG)150 repeat expansions. The mice exhibitprogressive neurological pathologies similar to HD, including, abnormaland involuntary movements, tremors, and epileptic seizures.

These transgenic mice show overt behavioral changes at approximately 8weeks of age. As early as 5 to 6 weeks of age, they display more subtledeficiencies in motor skills. Hsp70 inducing compounds of the inventioncan be administered by intravenous or oral administration at doses of10-100 mg per kg of body weight starting at various times (for example,at 5 to 6 weeks of age). Compounds can be given on multiple differentdosing schedules (e.g., once per week versus three times per week).Performance on one or more rodent motor tests such as swimming tank,beam walking, rotarod apparatus, and footprint test (see Carter, et al.,1999) can be performed to assess the activity of the compounds inpreventing loss of neurological function in HD mice.

Example 6 Parkinson's Disease (PD)

There are two widely employed models of PD in which disease is inducedby chemical treatment. These are the 6-OHDA (Zigmond, M. J. andStricker, E. M. (1984) Parkinson's disease: studies with an animalmodel. Life Sci. 35:5-18; Sauer, H, and Oertel, W. H. (1994) Progressivedegeneration of nigrostriatal dopamine neurons following intrastriatalterminal lesions with 6-hydroxydopamine: a combined retrograde tracingand immunocytochemical study in the rat. Neuroscience 59:401-415) andthe MPTP (Langston, J. W., Forno, L. S., Rebert, C. S., and Irwin, I.(1984) Selective nigral toxicity after systemic administration of1-methyl-4-phenyl-1,2,5,6-tetrahydropyrine (MPTP) in the squirrelmonkey. Brain Res. 292:390-4) models. An example of a test ofHsp70-inducing compounds of the invention using the 6-OHDA is described.

Young adult male rats are injected with Fluoro-Gold (FG) by stereotacticinjection into the striatum in the brain in order to facilitatevisualization of the neurons in the substantia nigra, the site of PD.Under anesthesia, 0.2 μl or a 4% solution of FG is administered bystereotactic injection (1 mm anterior from bregma, 3 mm lateral, and 4mm ventral from dura into both striata). One week after FG injection,the rats receive a stereotactic injection of 6-OHDA (20 μg dissolved in4 μl saline; Sigma) into the striatum on one side of the brain, at thesame coordinates as the FG injection. Hsp70 inducing compounds of theinvention can be administered by intravenous or oral administration atdoses of 10-100 mg per kg of body weight. The compounds can be given atthe time of 6-OHDA injection or some time (2-4 weeks, for example)subsequent to 6-OHDA treatment. Rats are sacrificed 8 and 16 weeks after6-OHDA injection. The endpoints of this model are 1) behavioral changesas monitored in-life at various times by assessment of turning(rotational) behavior using classical neurological read-out, and 2) thebrain is removed after sacrifice, thin sections are made using acryostat, and immunohistochemistry is performed as described in Zigmondand Stricker (1984). Efficacy of the Hsp70 inducing compounds of theinvention is demonstrated by a decrease in rotational behavior as wellas a reduction in the loss of nigral dopaminergic neurons.

Example 7 Alzheimer's Disease (AD)

There are several transgenic mouse models of AD. One such model that iswidely used to test the efficacy of drugs in AD was described byHolcomb, et al. (Holcomb, L., Gordon, M. N., McGowan, E., Yu, X.,Benkovic, S., Jantzen, P., Wright, K., Saad, L. Mueller, R., Morgan, D.,Sanders, S., Zehr, C., O'Campo, K., Hardy, J., Prada, C. M., Eckman, C.,Younkin, S., Hsiao, K., and Duff, K. (1998) Accelerated Alzheimer-typephenotype in transgenic mice carrying both mutant amyloid precursorprotein and presenilin 1 transgenes. Nature Medicine 4:97-100). Thismodel contains two different genes associated with AD. One is a mutationin the amyloid precursor protein (APP). The mutant APP (K670N, M671L)transgenic line, Tg2576, has elevated amyloid beta-protein levels at anearly age, and, later, develops extracellular AD-type A beta deposits inthe brain. The other gene is a mutated presenilin-1 (PS1) gene. Thedoubly transgenic progeny from a cross between Tg2576 and the PS1 mutantPS1M146L transgenic line develop large numbers of fibrillar A betadeposits in cerebral cortex and hippocampus far earlier than theirsingly transgenic Tg2576 mice.

Hsp70 inducing compounds of the invention can be dosed in mice atvarious times. The age of mice at the start of drug dosing may bevaried. For example, a treatment starting time may be at 3 months ofage, a time at which the brain deposits are first detectable. The dosemay be, for example, 10 to 100 mg/kg body weight administered once perweek or three times per week by the oral or intravenous route. Theeffect of drug treatment can be assessed by measuring AD-type depositsin the brain as well as by assessing function of the mice in a mazetest.

Example 8 Measurement of Heat Shock Protein 70 (Hsp70)

Plasma Hsp70 can be measured by a sandwich ELISA kit (StressgenBioreagents Victoria, British Columbia, CANADA) according to a modifiedprotocol in house. In brief, Hsp70 in plasma specimens and serialconcentrations of Hsp70 standard are captured onto 96-well plate onwhich anti-Hsp70 antibody was coated. Then captured Hsp70 is detectedwith a biotinylated anti-Hsp70 antibody followed by incubation witheuropium-conjugated streptavidin. After each incubation unboundmaterials are removed by washing. Finally, antibody-Hsp70 complex wasmeasured by time resolved fluorometry of europium. Concentration ofHsp70 is calculated from a standard curve.

Example 9 Measurement of Natural Killer Cell Cytotoxic Activity

The following procedure can be employed to assay NK cell activity in asubject. The procedure is adapted from Kantakamalakul W, Jaroenpool J,Pattanapanyasat K. A novel enhanced green fluorescent protein(EGFP)-K562 flow cytometric method for measuring natural killer (NK)cell cytotoxic activity. J Immunol Methods. 2003 Jan. 15: 272:189-197,the entire teachings of which are incorporated herein by reference.

Materials and methods: Human erythroleukaemic cell line, K562, isobtained from American Type Culture Collection (CCL-243, American TypeCulture Collection, Manassas, Va.), and cultured in RPMI-1640 medium(Cat#11875-093 Gibco Invitrogen Corp, Carlsbad, Calif.) supplementedwith 10% heat inactivated fetal calf serum (Gibco), 2 mM L-glutamine,100 μg/ml streptomycin and 100 IU/ml penicillin at 37° C. with 5% CO₂.K562 cells are transduced with retroviral vector which encode greenfluorescent protein (eGFP). Stable cell line is selected withantibiotic, G418. About 99.6% G418 resistant cells are eGFP positiveafter section.

The subject's peripheral blood mononuclear cells (PBMCs) are prepared byclinical study sites and received in BD Vacutainer Cell Preparation Tubewith sodium heparin (Product Number: 362753, Becton Dickinson, FranklinLakes, N.J.).

Two-fold serial dilution of 800 μl effector cells (patient's PBMC)starting at concentration of 1×10⁶ cells/mL are put into four individualpolystyrene 12×75-mm tubes. Log phase growing target cells (K562/eGFP)are adjusted with growth medium (RPMI-1640) to a concentration of 1×10⁵cells/mL and 100 μL targets then added into the tubes to provideeffector/target (E/T) ratios of 80:1, 40:1, 20:1, 10:1. Effector cellsalone and target cells alone are used as controls. All tubes areincubated at 37° C. with 5% CO₂ for about 3.5 hr. Ten microliters ofpropidium iodide (PI) at a concentration of 1 mg/mL is added to eachtube including effector and target control tubes and then incubated atroom temperature for 15 min.

Cytotoxic activity is analyzed with a FACSCalibur flow cytometer (BectonDickinson). Linear amplification of the Forward and side scatter(FSC/SSC) signals, as well as logarithmic amplification of eGFP and PIemission in green and red fluorescence is obtained. Ten thousand eventsper sample tube with no gating for acquisition are collected foranalysis. Data analysis for two-parameter dot plots for eGFP versus PIis performed using CELLQuest (Becton Dickinson Biosciences) software toenumerate live and dead target cells. Debris and dead cells are excludedby setting a threshold of forward light scatter.

Example 10 Inhibition of HUVEC Cell Migration

To examine if the compounds of the invention affect endothelial cellfunction, an in vitro human umbilical vein endothelial cell (HUVEC)migration assay is performed in the presence of a compound of theinvention. HUVEC cells (passage number 4) are cultured on 12-well platesand time-lapse imaging is performed with the live cell imaging system onan inverted microscope supplied with 6-7% CO₂. The temperature is keptat 37° C. Images are taken every 30 minutes using the 2× objective forup to 106 hr or every 60 seconds using the 20× objective for 30 min.Confluent HUVEC cultures are scraped similarly to make a blank area,followed by culturing in HUVEC medium for 15 hr without treatment. Themigration areas, which are imaged as time-lapse sequences for each well,are used as a basis to standardize/correct migration rates. Then,migration of cells under different treatments is imaged at the same timeto generate time-lapse image sequences for each well. Time-lapse moviesare further analyzed by measuring areas that are covered by migratingcells. During experiments, HUVEC cells are activated by the presence ofVEGF and basic FGF. Compounds of the invention (e.g. 100 nM and 1 μM)are expected to completely block migration of HUVEC cells to the blankarea, indicating that compounds of the invention possesses potentinhibitory effect on the migration of activated HUVEC cell in vitroinduced by VEGF and basic FGF.

It is also possible to track HUVEC behavior during above treatments. Itis expected that HUVEC cells will begin to shrink after 24 hr treatmentwith compounds of the invention.

Example 11 Enhanced VE-Cadherin Junctions of HUVEC Cells

An immunofluorescence study is performed by using anti-VE-cadherinantibodies to examine VE-cadherin junctions between HUVEC cells. HUVECcells are treated with DMSO or a compound of the invention (e.g. 10, 100and 1000 nM) for 24 hrs and fixed for immunostaining, DMSO concentrationis 1:100 for all treatments. To boost the immunofluorescence signal,cells are stained with a mixture of 2 polyclonal anti-human VE-cadherinAbs followed by staining with a mixture of fluorescent secondaryantibodies. It is expected that with compounds of the invention,VE-cadherin staining will be extremely strong in cell-cell junctionregions, but not the non-contacted regions compared to that in DMSOtreated cultures. Compounds of the invention are expected to enhance theassembly of cell-cell junctions of activated human endothelial cells,likely through induction of the accumulation of VE-cadherin molecules atthe junctions. This effect could result in limited motility of the cellsand reducing permeability of the endothelium, thus contributing to thecell migration inhibition and the potential anti-angiogenesis effect ofcompounds of the invention.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A compound comprising a bis[thiohydrazide amide]or a deprotonated form thereof complexed to a transition metal cation,wherein the bis[thiohydrazide amide] is represented by the followingStructural Formula:


2. The compound of claim 1, wherein the compound is greater than 90%pure by weight.
 3. The compound of claim 1, wherein the transition metalcation has a charge of +2.
 4. The compound of claim 3 wherein thetransition metal cation is Ni²⁺, Cu²⁺, Co²⁺, Fe²⁺, Zn²⁺, Pt²⁺ or Pd²⁺.5. The compound of claim 4, wherein the transition metal cation is Cu²⁺.6. The compound of claim 1, wherein the molar ratio of bis[thiohydrazideamide] or deprotonated form thereof to transition metal cation is equalto or greater than 0.5 and equal to or less than 2.0.
 7. The compound ofclaim 6, wherein the molar ratio of bis[thiohydrazide amide] ordeprotonated form thereof to transition metal cation is 1:1.
 8. Acompound represented by the following structural formula:

wherein X is a transition metal cation with a +2 charge.
 9. The compoundof claim 8, wherein the compound is greater than 90% pure by weight. 10.The compound of claim 9, wherein X is Ni²⁺, Cu²⁺, Co²⁺, Fe²⁺, Zn²⁺, Pt²⁺or Pd²⁺.
 11. The compound of claim 9, wherein X is Cu²⁺.
 12. Apharmaceutical composition comprising a compound and a pharmaceuticallyacceptable carrier or diluent, wherein the compound comprises abis[thiohydrazide amide] represented by the following StructuralFormula:

or a deprotonated form thereof complexed to a transition metal cation.13. The pharmaceutical composition of claim 12, wherein the transitionmetal cation is Ni²⁺, Cu²⁺, Co²⁺, Fe²⁺, Zn²⁺, Pt²⁺ or Pd²⁺.
 14. Thepharmaceutical composition of claim 12, wherein the molar ratio ofbis[thiohydrazide amide] or deprotonated form thereof to transitionmetal cation is equal to or greater than 0.5 and equal to or less than2.0.
 15. A pharmaceutical composition comprising a compound representedby the following Structural Formula:

and a pharmaceutical acceptable carrier or diluent, wherein X is atransition metal cation with a +2 charge.
 16. The pharmaceuticalcomposition of claim 15, wherein the transition metal cation is Ni²⁺,Cu²⁺, Co²⁺, Fe²⁺, Zn²⁺, Pt²⁺ or Pd²⁺.
 17. A method of treating a subjectwith melanoma, said method comprising administering to the subject aneffective, amount of a compound of claim
 1. 18. A method of treating asubject with melanoma, said method comprising administering to thesubject an effective amount of paclitaxel or a paclitaxel analog and aneffective amount of a compound of claim
 1. 19. A method of treating asubject with melanoma, said method comprising administering to thesubject an effective amount of a compound of claim
 8. 20. A method oftreating a subject with melanoma, said method comprising administeringto the subject an effective amount of paclitaxel or a paclitaxel analogand an effective amount of a compound of claim 8.